Δ12 desaturases suitable for altering levels of polyunsaturated fatty acids in oleaginous yeast

ABSTRACT

The present invention relates to fungal Δ12 fatty acid desaturases that are able to catalyze the conversion of oleic acid to linoleic acid (LA; 18:2). Nucleic acid sequences encoding the desaturases, nucleic acid sequences which hybridize thereto, DNA constructs comprising the desaturase genes, and recombinant host microorganisms expressing increased levels of the desaturases are described. Methods of increasing production of specific ω-3 and ω-6 fatty acids by over-expression of the Δ12 fatty acid desaturases are also described herein.

This application claims the benefit of U.S. Provisional Application No.60/519,191, filed Nov. 12, 2003, and U.S. Provisional Application No.60/570,679, filed May 13, 2004.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, thisinvention pertains to the identification of nucleic acid fragmentsencoding Δ12 fatty acid desaturase enzymes useful for disrupting orenhancing the production of polyunsaturated fatty acids in oleaginousmicroorganisms, such as oleaginous yeast.

BACKGROUND OF THE INVENTION

It has long been recognized that certain polyunsaturated fatty acids, orPUFAs, are important biological components of healthy cells. Forexample, such PUFAs are recognized as:

-   -   “Essential” fatty acids that can not be synthesized de novo in        mammals and instead must be obtained either in the diet or        derived by further desaturation and elongation of linoleic acid        (LA) or α-linolenic acid (ALA);    -   Constituents of plasma membranes of cells, where they may be        found in such forms as phospholipids or triglycerides;    -   Necessary for proper development, particularly in the developing        infant brain, and for tissue formation and repair; and,    -   Precursors to several biologically active eicosanoids of        importance in mammals, including prostacyclins, eicosanoids,        leukotrienes and prostaglandins.

In the 1970's, observations of Greenland Eskimos linked a low incidenceof heart disease and a high intake of long-chain ω-3 PUFAs (Dyerberg, J.et al., Amer. J. Clin Nutr. 28:958-966 (1975); Dyerberg, J. et al.,Lancet 2(8081):117-119 (Jul. 15, 1978)). More recent studies haveconfirmed the cardiovascular protective effects of ω-3 PUFAs (Shimokawa,H., Word Rev Nutr Diet, 88:100-108 (2001); von Schacky, C., andDyerberg, J., World Rev Nutr Diet, 88:90-99 (2001)). Further, it hasbeen discovered that several disorders respond to treatment with ω-3fatty acids, such as the rate of restenosis after angioplasty, symptomsof inflammation and rheumatoid arthritis, asthma, psoriasis and eczema.γ-Linolenic acid (GLA, an ω-6 PUFA) has been shown to reduce increasesin blood pressure associated with stress and to improve performance onarithmetic tests. GLA and dihomo-γ-linolenic acid (DGLA, another ω-6PUFA) have been shown to inhibit platelet aggregation, causevasodilation, lower cholesterol levels and inhibit proliferation ofvessel wall smooth muscle and fibrous tissue (Brenner et al., Adv. Exp.Med. Biol. 83: 85-101 (1976)). Administration of GLA or DGLA, alone orin combination with eicosapentaenoic acid (EPA, an ω-3 PUFA), has beenshown to reduce or prevent gastrointestinal bleeding and other sideeffects caused by non-steroidal anti-inflammatory drugs (U.S. Pat. No.4,666,701). Further, GLA and DGLA have been shown to prevent or treatendometriosis and premenstrual syndrome (U.S. Pat. No. 4,758,592) and totreat myalgic encephalomyelitis and chronic fatigue after viralinfections (U.S. Pat. No. 5,116,871). Other evidence indicates thatPUFAs may be involved in the regulation of calcium metabolism,suggesting that they may be useful in the treatment or prevention ofosteoporosis and kidney or urinary tract stones. Finally, PUFAs can beused in the treatment of cancer and diabetes (U.S. Pat. No. 4,826,877;Horrobin et al., Am. J. Clin. Nutr. 57 (Suppl.): 732S-737S (1993)).

PUFAs are generally divided into two major classes (consisting of theω-6 and the ω-3 fatty acids) that are derived by desaturation andelongation of the essential fatty acids, LA and ALA, respectively.Despite a variety of commercial sources of PUFAs from natural sources[e.g., seeds of evening primrose, borage and black currants; filamentousfungi (Mortierella), Porphyridium (red alga), fish oils and marineplankton (Cyclotella, Nitzschia, Crypthecodinium)], there are severaldisadvantages associated with these methods of production. First,natural sources such as fish and plants tend to have highlyheterogeneous oil compositions. The oils obtained from these sourcestherefore can require extensive purification to separate or enrich oneor more of the desired PUFAs. Natural sources are also subject touncontrollable fluctuations in availability (e.g., due to weather,disease, or over-fishing in the case of fish stocks); and, crops thatproduce PUFAs often are not competitive economically with hybrid cropsdeveloped for food production. Large-scale fermentation of someorganisms that naturally produce PUFAs (e.g., Porphyridium, Mortierella)can also be expensive and/or difficult to cultivate on a commercialscale.

As a result of the limitations described above, extensive work has beenconducted toward: 1.) the development of recombinant sources of PUFAsthat are easy to produce commercially; and 2.) modification of fattyacid biosynthetic pathways, to enable production of desired PUFAs. Forexample, advances in the isolation, cloning and manipulation of fattyacid desaturase and elongase genes from various organisms have been madeover the last several years. Knowledge of these gene sequences offersthe prospect of producing a desired fatty acid and/or fatty acidcomposition in novel host organisms that do not naturally produce PUFAs.The literature reports a number of examples in Saccharomyces cerevisiae,such as: Domergue, F., et al. (Eur. J. Biochem. 269:4105-4113 (2002)),wherein two desaturases from the marine diatom Phaeodactylum tricomutumwere cloned into S. cerevisiae, leading to the production of EPA;Beaudoin F., et al. (Proc. Natl. Acad. Sci. U.S.A. 97(12):6421-6(2000)), wherein the ω-3 and ω-6 PUFA biosynthetic pathways werereconstituted in S. cerevisiae, using genes from Caenorhabditis elegans;Dyer, J. M., et al. (Appl. Eniv. Microbiol., 59:224-230 (2002)), whereinplant fatty acid desaturases (FAD2 and FAD3) were expressed in S.cerevisiae, leading to the production of ALA; and, U.S. Pat. No.6,136,574 (Knutzon et al., Abbott Laboratories), wherein one desaturasefrom Brassica napus and two desaturases from the fungus Mortierellaalpina were cloned into S. cerevisiae, leading to the production of LA,GLA, ALA and STA. There remains a need, however, for an appropriatemicrobial system in which these types of genes can be expressed toprovide for economical production of commercial quantities of one ormore PUFAs. Additionally, a need exists for oils enriched in specificPUFAs, notably EPA and DHA.

One class or microorganisms that has not been previously examined as aproduction platform for PUFAs are the oleaginous yeast. These organismscan accumulate oil up to 80% of their dry cell weight. The technologyfor growing oleaginous yeast with high oil content is well developed(for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol.16:119-206 (1982)), and may offer a cost advantage compared tocommercial micro-algae fermentation for production of ω-3- or ω-6 PUFAs.Whole yeast cells may also represent a convenient way of encapsulatingω-3 or ω-6 PUFA-enriched oils for use in functional foods and animalfeed supplements.

Despite the advantages noted above, most oleaginous yeast are naturallydeficient in ω-6 and ω-3 PUFAs, since naturally produced PUFAs in theseorganisms are usually limited to 18:2 fatty acids (and less commonly,18:3 fatty acids). Thus, the problem to be solved is to develop anoleaginous yeast that accumulates oils enriched in ω-3 and/or ω-6 fattyacids. Toward this end, it is not only necessary to introduce therequired desaturases and elongases that allow for the synthesis andaccumulation of ω-3 and/or ω-6 fatty acids in oleaginous yeast, but alsoto increase the availability of the 18:2 substrate (i.e., LA).Generally, the availability of this substrate is controlled by theactivity of Δ12 desaturases that catalyze the conversion of oleic acidto LA.

There are a variety of known Δ12 desaturases disclosed in the publicliterature, some of which originate from fungal sources (e.g.,Mortierella alpina, Emericella nidulans, Mucor rouxii). Thesedesaturases are not known to be effective for altering fatty acidcomposition in oleaginous yeast, although the Mortierella alpinadesaturase, for example, has previously been expressed in thenon-oleaginous yeast Saccharomyces cerevisiae and enabled accumulationof 18:2 (Sakuradani E., et al., Eur J Biochem. 261 (3):812-20 (1999)).WO 2003/099216 describes Δ12 desaturases from Neurospora crassa andBotrytis cinerea. Subsequent expression analysis in S. cerevisiaeconfirmed the ability of the N. crassa desaturase to convert oleic acidto 18:2; however, the percent substrate conversion for this reaction wasonly 68% (calculated as ([18:2]/[18:1+18:2])*100). Thus, there is needfor the identification and isolation of genes encoding Δ12 desaturasesthat are able to support production of high levels of 18:2 (LA) inoil-producing host organisms (e.g., oleaginous yeast) for use in theproduction of PUFAs.

Applicants have solved the stated problem by isolating the gene encodinga Δ12 desaturase from the fungus Fusarium moniliforme and demonstratingsurprisingly efficient conversion of oleic acid to 18:2 (LA) uponexpression in an oleaginous yeast. Furthermore, orthologs of this Δ12desaturase have been identified in Aspergillus nidulans, Aspergillusflavus, Aspergillus fumigatus, Magnaporthe grisea, Neurospora crassa andFusarium graminearium.

SUMMARY OF THE INVENTION

The invention relates to a gene encoding a Δ12 desaturase enzymeisolated from Fusarium useful for the manipulation of the biochemicalpathway leading to the production of ω-3 and ω-6 fatty acids.Accordingly, the invention provides an isolated nucleic acid fragmentencoding a fungal Δ12 desaturase enzyme, selected from the groupconsisting of:

-   -   (a) an isolated nucleic acid fragment encoding the amino acid        sequence as set forth in SEQ ID NO:4;    -   (b) an isolated nucleic acid fragment that hybridizes with (a)        under the following hybridization conditions: 0.1×SSC, 0.1% SDS,        65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1%        SDS; or    -   (c) an isolated nucleic acid fragment that is complementary        to (a) or (b).

In one specific embodiment the invention provides an isolated nucleicacid fragment comprising a first nucleotide sequence, encoding a Δ12desaturase enzyme, having at least 89.2% identity based on the Clustalmethod of alignment when compared to the nucleic acid fragment havingthe sequence as set forth in SEQ ID NO:3;

-   -   or a second nucleotide sequence comprising the complement of the        first nucleotide sequence.

Similarly the invention provides an isolated nucleic acid fragmentcomprising a first nucleotide sequence encoding a Δ12 desaturase enzymeof at least 477 amino acids that has at least 95% identity based on theClustal method of alignment when compared to a polypeptide having thesequence as set forth in SEQ ID NO:4;

-   -   or a second nucleotide sequence comprising the complement of the        first nucleotide sequence.

Similarly the invention provides polypeptides encoded by the isolatednucleic acids of the invention as well as genetic chimera of thesenucleic acids and transformed host cells comprising the same.

In another embodiment the invention provides a method of obtaining anucleic acid fragment encoding a Δ12 desaturase enzyme comprising:

-   -   (a) probing a genomic library with the nucleic acid fragment of        the invention;    -   (b) identifying a DNA clone that hybridizes with the nucleic        acid fragment of the invention; and    -   (c) sequencing the genomic fragment that comprises the clone        identified in step (b),

wherein the sequenced genomic fragment encodes a Δ12 desaturase enzyme.

Similarly the invention provides a method of obtaining a nucleic acidfragment encoding a Δ12 desaturase enzyme comprising:

-   -   (a) synthesizing at least one oligonucleotide primer        corresponding to a portion of the sequence as set forth in SEQ        ID NOs:4, 8, 12, 16, 20, 21 and 22; and    -   (b) amplifying an insert present in a cloning vector using the        oligonucleotide primer of step (a);        wherein the amplified insert encodes a portion of an amino acid        sequence encoding a Δ12 desaturase enzyme.

In another embodiment the invention provides a method for producinglinoleic acid comprising:

-   -   a) providing an oleaginous yeast comprising:        -   (i) an isolated nucleic acid fragment encoding a fungal            polypeptide having Δ12 desaturase activity that has at least            56.3% identity based on the Clustal method of alignment when            compared to a polypeptide having the sequence as set forth            in SEQ ID NO:4; and        -   (ii) a source of oleic acid;    -   b) growing the yeast of step (a) under conditions wherein the        chimeric desaturase gene is expressed and the oleic acid is        converted to linoleic acid; and    -   c) optionally recovering the linoleic acid of step (b).

Similarly the invention provides a method for the production of ω-3 orω-6 polyunsaturated fatty acids comprising:

-   -   a) providing an oleaginous yeast comprising:        -   (i) an isolated nucleic acid fragment encoding a protein            having Δ12 desaturase activity that has at least 56.3%            identity based on the Clustal method of alignment when            compared to a polypeptide having the sequence as set forth            in SEQ ID NO:4; and        -   (ii) genes encoding a functional ω-3/ω-6 fatty acid            biosynthetic pathway;    -   b) providing a source of desaturase substrate comprising oleic        acid; and    -   c) contacting the oleaginous yeast of (a) with the desaturase        substrate of (b) wherein polyunsaturated fatty acids are        produced; and    -   d) optionally recovering the polyunsaturated fatty acids of step        (c).

Additionally the invention provides microbial oils produced by themethods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIG. 1 shows a schematic illustration of the biochemical mechanism forlipid accumulation in oleaginous yeast.

FIG. 2 illustrates the ω-3 and ω-6 fatty acid biosynthetic pathways.

FIG. 3 illustrates the construction of the plasmid vector pY5 for geneexpression in Yarrowia lipolytica.

FIG. 4 shows a phylogenetic tree of proteins from different filamentousfungi (i.e., Aspergillus nidulans, Fusarium moniliforme, F.graminearium, Magnaporthe grisea and Neurospora crassa) having homologyto the Yarrowia lipolytica Δ12 desaturase enzyme, and created usingMegalign DNASTAR software.

FIG. 5 shows a pairwise comparison (% Identity) between and amongproteins from different filamentous fungi (i.e., Aspergillus nidulans,Fusarium moniliforme, F. graminearium, Magnaporthe grisea and Neurosporacrassa) having homology to the Yarrowia lipolytica Δ12 desaturase enzymeusing a ClustalW analysis (Megalign program of DNASTAR sofware).

FIG. 6 shows a pairwise comparison (% Identity) between proteins fromdifferent filamentous fungi having homology to the Yarrowia lipolyticaΔ12 desaturase and Δ12 desaturase proteins from some other fungal andnon-fungal species using a ClustalW analysis (Megalign program ofDNASTAR sofware).

FIG. 7 provides a plasmid map for pKUNF12T6E.

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NOs:1-22, 51 and 52 are ORFs encoding genes or proteins asidentified in Table 1.

TABLE 1 Summary Of Desaturase Gene And Protein SEQ ID Numbers ORFNucleic acid Protein Description SEQ ID NO. SEQ ID NO. Fusariummoniliforme sub-family 1  1  2 desaturase (Δ15/Δ12 desaturase) (1209 bp)(402 AA) Fusarium moniliforme sub-family 2  3  4 desaturase (Δ12desaturase) (1434 bp) (477 AA) Aspergillus nidulans sub-family 1  5  6desaturase (Δ15 desaturase) (1206 bp) (401 AA) Aspergillus nidulanssub-family 2  7  8 desaturase (Δ12 desaturase) (1416 bp) (471 AA)Magnaporthe grisea sub-family 1  9 10 desaturase (Δ15 desaturase) (1185bp) (394 AA) Magnaporthe grisea sub-family 2 11 12 desaturase (Δ12desaturase) (1656 bp) (551 AA) Neurospora crassa sub-family 1 13 14desaturase (Δ15 desaturase) (1290 bp) (429 AA) Neurospora crassasub-family 2 15 16 desaturase (Δ12 desaturase) (1446 bp) (481 AA)Fusarium graminearium sub-family 1 17 18 desaturase (Δ15 desaturase)(1212 bp) (403 AA) Fusarium graminearium sub-family 2 19 20 desaturase(Δ12 desaturase) (1371 bp) (456 AA) Aspergillus fumigatus sub-family 2 —21 desaturase (Δ12 desaturase) (424 AA) Aspergillus flavus sub-family 2— 22 desaturase (Δ12 desaturase) (466 AA) Yarrowia lipolytica Δ12desaturase 51 52 (1936 bp) (419 AA)

SEQ ID NOs:23 and 24 are primers TEF 5′ and TEF 3′, respectively, usedto isolate the TEF promoter.

SEQ ID NOs:25 and 26 are primers XPR 5′ and XPR 3′, respectively, usedto isolate the XPR2 transcriptional terminator.

SEQ ID NOs:27-38 correspond to primers YL5, YL6, YL9, YL10, YL7, YL8,YL3, YL4, YL1, YL2, YL61 and YL62, respectively, used for plasmidconstruction.

SEQ ID NOs:39 and 41 are the degenerate primers identified as P73 andP76, respectively, used for the isolation of a Yarrowia lipolytica Δ12desaturase gene.

SEQ ID NOs:40 and 42 are the amino acid consensus sequences thatcorrespond to the degenerate primers P73 and P76, respectively.

SEQ ID NOs:43-46 correspond to primers P99, P100, P101 and P102,respectively, used for targeted disruption of the native Y. lipolyticaΔ12 desaturase gene.

SEQ ID NOs:47-50 correspond to primers P119, P120, P121 and P122,respectively, used to screen for targeted integration of the disruptedY. lipolytica Δ12 desaturase gene.

SEQ ID NOs:53 and 54 correspond to primers P147 and P148, respectively,used to amplify the full-length Y. lipolytica Δ12 desaturase codingregion.

SEQ ID NOs:55 and 56 correspond to primers P194 and P195, respectively,used to amplify the full-length Fusarium moniliforme Δ12 desaturasecoding region.

SEQ ID NO:57 provides the DNA sequence of plasmid pKUNF12T6E.

SEQ ID NO:58 corresponds to the Yarrowia lipolytica FBAIN promoterregion.

SEQ ID NO:59 is the 957 bp nucleotide sequence of a synthetic elongase 1gene derived from Mortierella alpina, codon-optimized for expression inY. lipolytica, while SEQ ID NO:60 is the corresponding 318 amino acidsequence.

SEQ ID NO:61 is the 1374 bp nucleotide sequence of a synthetic Δ6desaturase gene derived from Mortierella alpina, codon-optimized forexpression in Y. lipolytica, while SEQ ID NO:62 is the corresponding 457amino acid sequence.

SEQ ID NO:63 corresponds to the Yarrowia lipolytica FBA promoter region.

SEQ ID NO:64 is the 819 bp nucleotide sequence of a synthetic elongase 2gene derived from Thraustochytrium aureum, codon-optimized forexpression in Y. lipolytica, while SEQ ID NO:65 is the corresponding 272amino acid sequence.

SEQ ID NO:66 corresponds to the codon-optimized translation initiationsite for genes optimally expressed in Yarrowia sp.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, Applicants have isolated andconfirmed the identity of a Fusarium moniliforme gene encoding a Δ12desaturase and identified its orthologs in other fungi. Additionally,methods and compositions are provided which permit modification of thelong-chain polyunsaturated fatty acid (PUFA) content and composition ofoleaginous yeast, such as Yarrowia lipolytica.

The invention relates to novel Δ12 desaturase enzymes and genes encodingthe same that may be used for the manipulation of biochemical pathwaysfor the production of healthful PUFAs. Thus, the subject invention findsmany applications. PUFAs, or derivatives thereof, made by themethodology disclosed herein can be used as dietary substitutes, orsupplements, particularly infant formulas, for patients undergoingintravenous feeding or for preventing or treating malnutrition.Alternatively, the purified PUFAs (or derivatives thereof) may beincorporated into cooking oils, fats or margarines formulated so that innormal use the recipient would receive the desired amount for dietarysupplementation. The PUFAs may also be incorporated into infantformulas, nutritional supplements or other food products and may finduse as anti-inflammatory or cholesterol lowering agents. Optionally, thecompositions may be used for pharmaceutical use (human or veterinary).In this case, the PUFAs are generally administered orally but can beadministered by any route by which they may be successfully absorbed,e.g., parenterally (e.g., subcutaneously, intramuscularly orintravenously), rectally, vaginally or topically (e.g., as a skinointment or lotion).

Supplementation of humans or animals with PUFAs produced by recombinantmeans can result in increased levels of the added PUFAs, as well astheir metabolites. For example, treatment with arachidonic acid (ARA)can result not only in increased levels of ARA, but also downstreamproducts of ARA such as prostaglandins. Complex regulatory mechanismscan make it desirable to combine various PUFAs, or add differentconjugates of PUFAs, in order to prevent, control or overcome suchmechanisms to achieve the desired levels of specific PUFAs in anindividual.

definitions

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).

The term “Fusarium moniliforme” is synonymous with “Fusariumverticillioides”.

The term “fatty acids” refers to long-chain aliphatic acids (alkanoicacids) of varying chain length, from about C₁₂ to C₂₂ (although bothlonger and shorter chain-length acids are known). The predominant chainlengths are between C₁₆ and C₂₂. The structure of a fatty acid isrepresented by a simple notation system of “X:Y”, where X is the totalnumber of carbon atoms in the particular fatty acid and Y is the numberof double bonds.

Generally, fatty acids are classified as saturated or unsaturated. Theterm “saturated fatty acids” refers to those fatty acids that have no“double bonds” between their carbon backbone. In contrast, “unsaturatedfatty acids” have “double bonds” along their carbon backbones (which aremost commonly in the cis-configuration). “Monounsaturated fatty acids”have only one “double bond” along the carbon backbone (e.g., usuallybetween the 9^(th) and 10^(th) carbon atom as for palmitoleic acid(16:1) and oleic acid (18:1)), while “polyunsaturated fatty acids” (or“PUFAs”) have at least two double bonds along the carbon backbone (e.g.,between the 9^(th) and 10^(th), and 12^(th) and 13^(th) carbon atoms forlinoleic acid (18:2); and between the 9^(th) and 10^(th), 12^(th) and13^(th), and 15^(th) and 16^(th) for α-linolenic acid (18:3)).

“PUFAs” can be classified into two major families (depending on theposition (n) of the first double bond nearest the methyl end of thefatty acid carbon chain). Thus, the “omega-6 fatty acids” (ω-6 or n-6)have the first unsaturated double bond six carbon atoms from the omega(methyl) end of the molecule and additionally have a total of two ormore double bonds, with each subsequent unsaturation occurring 3additional carbon atoms toward the carboxyl end of the molecule. Incontrast, the “omega-3 fatty acids” (ω-3 or n-3) have the firstunsaturated double bond three carbon atoms away from the omega end ofthe molecule and additionally have a total of three or more doublebonds, with each subsequent unsaturation occurring 3 additional carbonatoms toward the carboxyl end of the molecule.

For the purposes of the present disclosure, the omega-reference systemwill be used to indicate the number of carbons, the number of doublebonds and the position of the double bond closest to the omega carbon,counting from the omega carbon (which is numbered 1 for this purpose).This nomenclature is shown below in Table 2, in the column titled“Shorthand Notation”. The remainder of the Table summarizes the commonnames of (ω-3 and ω-6 fatty acids, the abbreviations that will be usedthroughout the specification, and each compounds' chemical name.

TABLE 2 Nomenclature Of Polyunsaturated Fatty Acids Shorthand CommonName Abbreviation Chemical Name Notation Linoleic LA cis-9,12-octadecadienoic 18:2 ω-6 γ-Linolenic GLA cis-6, 9, 12- 18:3 ω-6octadecatrienoic Eicosadienoic EDA cis-11, 14- eicosadienoic 20:2 ω-6Dihomo-γ- DGLA cis-8, 11, 14- 20:3 ω-6 Linoleic eicosatrienoicArachidonic ARA cis-5, 8, 11, 14- 20:4 ω-6 eicosatetraenoic α-LinolenicALA cis-9, 12, 15- 18:3 ω-3 octadecatrienoic Stearidonic STA cis-6, 9,12, 15- 18:4 ω-3 octadecatetraenoic Eicosatrienoic ETrA cis-11, 14, 17-20:3 ω-3 eicosatrienoic Eicosatetraenoic ETA cis-8, 11, 14, 17- 20:4 ω-3eicosatetraenoic Eicosapentaenoic EPA cis-5, 8, 11, 14, 17- 20:5 ω-3eicosapentaenoic Docosapentaenoic DPA cis-7, 10, 13, 16, 19- 22:5 ω-3docosapentaenoic Docosahexaenoic DHA cis-4, 7, 10, 13, 16, 19- 22:6 ω-3docosahexaenoic

The term “essential fatty acid” refers to a particular PUFA that anindividual must ingest in order to survive, being unable to synthesizethe particular essential fatty acid de novo. Linoleic (18:2, ω-6) andlinoleic (18:3, ω-3) fatty acids are “essential fatty acids”, sincehumans cannot synthesize them and have to obtain them in their diet.

The term “fat” refers to a lipid substance that is solid at 25° C. andusually saturated.

The term “oil” refers to a lipid substance that is liquid at 25° C. andusually polyunsaturated. PUFAs are found in the oils of some algae,oleaginous yeast and filamentous fungi. “Microbial oils” or “single celloils” are those oils naturally produced by microorganisms during theirlifespan. Such oils can contain long-chain PUFAs.

The term “PUFA biosynthetic pathway enzyme” refers to any of thefollowing enzymes (and genes which encode said enzymes) associated withthe biosynthesis of a PUFA, including: a Δ4 desaturase, a Δ5 desaturase,a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, aΔ9 desaturase, a Δ8 desaturase and/or an elongase(s).

The term “ω-3/ω-6 fatty acid biosynthetic pathway” refers to a set ofgenes which, when expressed under the appropriate conditions encodeenzymes that catalyze the production of either or both ω-3 and ω-6 fattyacids. Typically the genes involved in the ω-3/ω-6 fatty acidbiosynthetic pathway encode some or all of the following enzymes: Δ12desaturase, Δ6 desaturase, elongase, Δ5 desaturase, Δ17 desaturase, Δ15desaturase, Δ9 desaturase, Δ8 desaturase and Δ4 desaturase. Arepresentative pathway is illustrated in FIG. 2, providing for theconversion of oleic acid through various intermediates to DHA, whichdemonstrates how both ω-3 and ω-6 fatty acids may be produced from acommon source. The pathway is naturally divided into two portions whereone portion will generate ω-3 fatty acids and the other portion, onlyω-6 fatty acids. That portion that only generates ω-3 fatty acids willbe referred to herein as the ω-3 fatty acid biosynthetic pathway,whereas that portion that generates only ω-6 fatty acids will bereferred to herein as the ω-6 fatty acid biosynthetic pathway.

The term “functional” as used herein in context with the ω-3/ω-6 fattyacid biosynthetic pathway means that some (or all of) the genes in thepathway express active enzymes. It should be understood that “ωc-3/ω-6fatty acid biosynthetic pathway” or “functional ω-3/ω-6 fatty acidbiosynthetic pathway” does not imply that all the genes listed in theabove paragraph are required, as a number of fatty acid products willonly require the expression of a subset of the genes of this pathway.

The term “desaturase” refers to a polypeptide that can desaturate, i.e.,introduce a double bond, in one or more fatty acids to produce a mono-or polyunsaturated fatty acid. Despite use of the omega-reference systemthroughout the specification in reference to specific fatty acids, it ismore convenient to indicate the activity of a desaturase by countingfrom the carboxyl end of the substrate using the delta-system. Ofparticular interest herein are Δ12 desaturases that desaturate a fattyacid between the 12^(th) and 13^(th) carbon atoms numbered from thecarboxyl-terminal end of the molecule and that catalyze the conversionof oleic acid to LA. Other desaturases relevant to the presentdisclosure include: Δ15 desaturases that catalyze the conversion of LAto ALA; Δ17 desaturases that catalyze the conversion of DGLA to ETAand/or ARA to EPA; Δ6 desaturases that catalyze the conversion of LA toGLA and/or ALA to STA; Δ5 desaturases that catalyze the conversion ofDGLA to ARA and/or ETA to EPA; Δ4 desaturases that catalyze theconversion of DPA to DHA; Δ8 desaturases that catalyze the conversion ofEDA to DGLA and/or ETrA to ETA; and Δ9 desaturases that catalyze theconversion of palmitate to palmitoleic acid (16:1) and/or stearate tooleic acid (18:1). In the art, Δ15 and Δ17 desaturases are alsooccassionally referred to as “omega-3 desaturases”, “w-3 desaturases”,and/or “ω-3 desaturases”. Some desaturases have activities on two ormore substrates (e.g., the substrates of the Saprolegnia diclina Δ17desaturase include ARA and DGLA, while those of the Caenorhabditiselegans ω-3 desaturase include LA and GLA).

The term “proteins having homology to the Yarrowia lipolytica Δ12desaturase” refers to the proteins identified herein as SEQ ID NOs:2, 4,6, 8, 10, 12, 14, 16, 18, 20, 21 and 22, and that have homology to theY. lipolytica desaturase identified herein as SEQ ID NO:52(characterized in co-pending U.S. patent application Ser. No.10/840,325, herein incorporated by reference in its entirety).Phylogenetic analysis determined that these proteins (i.e., SEQ IDNOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 21 and 22) clustered into twodistinct sub-families, referred to herein as “Sub-family 1” and“Sub-family 2”. Specifically, the Sub-family 1 proteins appear to encodeΔ15 desaturases (i.e., SEQ ID NOs:2, 6, 10, 14 and 18; see co-pendingU.S. Provisional Application 60/519,191, herein incorporated byreference in its entirety). In contrast, the Sub-family 2 proteinsencode proteins with Δ12 desaturase activity (i.e., SEQ ID NOs:4, 8, 12,16, 20, 21 and 22) as characterized herein.

The terms “conversion efficiency” and “percent substrate conversion”refer to the efficiency by which a particular enzyme (e.g., a desaturaseor elongase) can convert substrate to product. The conversion efficiencyis measured according to the following formula:([product]/[substrate+product])*100, where ‘product’ includes theimmediate product and all products in the pathway derived from it. Inthe present Application, it is desirable to identify those Δ12desaturases characterized by a high percent substrate conversion whenexpressed in oleaginous yeast hosts; thus, for example, a conversionefficiency to LA of at least about 70% is preferred, while a conversionefficiency to LA of at least about 80% is particularly suitable, and aconversion efficiency to LA of at least about 85% is most preferred.

The term “elongase” refers to a polypeptide that can elongate a fattyacid carbon chain to produce an acid that is 2 carbons longer than thefatty acid substrate that the elongase acts upon. This process ofelongation occurs in a multi-step mechanism in association with fattyacid synthase, whereby CoA is the acyl carrier (Lassner et al., ThePlant Cell 8:281-292 (1996)). Briefly, malonyl-CoA is condensed with along-chain acyl-CoA to yield CO₂ and a β-ketoacyl-CoA (where the acylmoiety has been elongated by two carbon atoms). Subsequent reactionsinclude reduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA, anda second reduction to yield the elongated acyl-CoA. Examples ofreactions catalyzed by elongases are the conversion of GLA to DGLA, STAto ETA, and EPA to DPA. Accordingly, elongases can have differentspecificities. For example, a C_(16/18) elongase will prefer a C₁₆substrate, a C_(18/20) elongase will prefer a C₁₈ substrate and aC_(20/22) elongase will prefer a C₂₀ substrate. In like manner, a Δ9elongase is able to catalyze the conversion of LA and ALA to EDA andETrA, respectively.

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of lipid (Weete, In: Fungal LipidBiochemistry, 2^(nd) ed., Plenum, 1980). These include oilseed plants(e.g., soybean, corn, safflower, sunflower, canola, rapeseed, flax,maize and primrose) and microorganisms (e.g., Thraustochytrium sp.,Schizochytrium sp., Mortierella sp. and certain oleaginous yeast).

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that can make oil. Generally, the cellular oil or triacylglycerolcontent of oleaginous microorganisms follows a sigmoid curve, whereinthe concentration of lipid increases until it reaches a maximum at thelate logarithmic or early stationary growth phase and then graduallydecreases during the late stationary and death phases (Yongmanitchai andWard, Appl. Environ. Microbiol. 57:419-25 (1991)). It is not uncommonfor oleaginous microorganisms to accumulate in excess of about 25% oftheir dry cell weight as oil. Examples of oleaginous yeast include, butare no means limited to, the following genera: Yarrowia, Candida,Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

The term “fermentable carbon substrate” means a carbon source that amicroorganism will metabolize to derive energy. Typical carbonsubstrates of the invention include, but are not limited to:monosaccharides, oligosaccharides, polysaccharides, alkanes, fattyacids, esters of fatty acids, monoglycerides, carbon dioxide, methanol,formaldehyde, formate and carbon-containing amines.

The term “codon optimized” as it refers to genes or coding regions ofnucleic acid fragments for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide for which the DNA codes.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A nucleic acid fragment is “hybridizable” to another nucleic acidfragment, such as a cDNA, genomic DNA, or RNA molecule, when asingle-stranded form of the nucleic acid fragment can anneal to theother nucleic acid fragment under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989),particularly Chapter 11 and Table 11.1 therein (entirely incorporatedherein by reference). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Stringency conditionscan be adjusted to screen for moderately similar fragments (such ashomologous sequences from distantly related organisms), to highlysimilar fragments (such as genes that duplicate functional enzymes fromclosely related organisms). Post-hybridization washes determinestringency conditions. One set of preferred conditions uses a series ofwashes starting with 6×SSC, 0.5% SDS at room temperature for 15 min,then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and thenrepeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A morepreferred set of stringent conditions uses higher temperatures in whichthe washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C.Another preferred set of highly stringent conditions uses two finalwashes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringentconditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washedwith 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridizations with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). In one embodimentthe length for a hybridizable nucleic acid is at least about 10nucleotides. Preferably a minimum length for a hybridizable nucleic acidis at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least about 30nucleotides. Furthermore, the skilled artisan will recognize that thetemperature and wash solution salt concentration may be adjusted asnecessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993)).In general, a sequence of ten or more contiguous amino acids or thirtyor more nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to specifically identify and/or isolatea nucleic acid fragment comprising the sequence. The instantspecification teaches the complete amino acid and nucleotide sequenceencoding particular fungal proteins. The skilled artisan, having thebenefit of the sequences as reported herein, may now use all or asubstantial portion of the disclosed sequences for purposes known tothose skilled in this art. Accordingly, the instant invention comprisesthe complete sequences as reported in the accompanying Sequence Listing,as well as substantial portions of those sequences as defined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine. Accordingly, the instant inventionalso includes isolated nucleic acid fragments that are complementary tothe complete sequences as reported in the accompanying Sequence Listing,as well as those substantially similar nucleic acid sequences.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: N.J. (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: N.Y. (1991). Preferred methods to determine identity aredesigned to give the best match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Sequence alignments and percent identity calculationsmay be performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences is performed using the Clustal method ofalignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10), unless otherwisespecified. Default parameters for pairwise alignments using the Clustalmethod are: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

Suitable nucleic acid fragments (isolated polynucleotides of the presentinvention) encode polypeptides that are at least about 70% identical,preferably at least about 75% identical, and more preferably at leastabout 80% identical to the amino acid sequences reported herein.Preferred nucleic acid fragments encode amino acid sequences that areabout 85% identical to the amino acid sequences reported herein. Morepreferred nucleic acid fragments encode amino acid sequences that are atleast about 90% identical to the amino acid sequences reported herein.Most preferred are nucleic acid fragments that encode amino acidsequences that are at least about 95% identical to the amino acidsequences reported herein. Suitable nucleic acid fragments not only havethe above homologies but typically encode a polypeptide having at least50 amino acids, preferably at least 100 amino acids, more preferably atleast 150 amino acids, still more preferably at least 200 amino acids,and most preferably at least 250 amino acids.

The term “homology” refers to the relationship among sequences wherebythere is some extent of likeness, typically due to descent from a commonancestral sequence. Homologous sequences can share homology based ongenic, structural, functional and/or behavioral properties. The term“ortholog” or “orthologous sequences” refers herein to a relationshipwhere sequence divergence follows speciation (i.e., homologous sequencesin different species arose from a common ancestral gene duringspeciation). In contrast, the term “paralogous” refers to homologoussequences within a single species that arose by gene duplication. Oneskilled in the art will be familiar with techniques required to identifyhomologous, orthologous and paralogous sequences.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Chemically synthesized”, as related to a sequence of DNA, means thatthe component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures;or automated chemical synthesis can be performed using one of a numberof commercially available machines. “Synthetic genes” can be assembledfrom oligonucleotide building blocks that are chemically synthesizedusing procedures known to those skilled in the art. These buildingblocks are ligated and annealed to form gene segments that are thenenzymatically assembled to construct the entire gene. Accordingly, thegenes can be tailored for optimal gene expression based on optimizationof nucleotide sequence to reflect the codon bias of the host cell. Theskilled artisan appreciates the likelihood of successful gene expressionif codon usage is biased towards those codons favored by the host.Determination of preferred codons can be based on a survey of genesderived from the host cell, where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, and that may refer to the coding region alone or may includeregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” gene refers to a gene that is introduced intothe host organism by gene transfer. Foreign genes can comprise nativegenes introduced into a non-native organism, native genes introducedinto a new location within the native host, or chimeric genes. A“transgene” is a gene that has been introduced into the genome by atransformation procedure. A “codon-optimized gene” is a gene having itsfrequency of codon usage designed to mimic the frequency of preferredcodon usage of the host cell.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites andstem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The terms “3′ non-coding sequences” and “transcription terminator” referto DNA sequences located downstream of a coding sequence. This includespolyadenylation recognition sequences and other sequences encodingregulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor. The 3′ region can influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” or “mRNA” refersto the RNA that is without introns and that can be translated intoprotein by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to, and derived from, mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065; WO99/28508). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, or the coding sequence. “Functional RNA” refers toantisense RNA, ribozyme RNA, or other RNA that is not translated and yethas an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment(s) of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be (but are not limited to)intracellular localization signals.

“Transformation” refers to the transfer of a nucleic acid fragment intoa host organism, resulting in genetically stable inheritance. Thenucleic acid fragment may be a plasmid that replicates autonomously, forexample; or, it may integrate into the genome of the host organism. Hostorganisms containing the transformed nucleic acid fragments are referredto as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing e.g., a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The term “homologous recombination” refers to the exchange of DNAfragments between two DNA molecules (during cross over). The fragmentsthat are exchanged are flanked by sites of identical nucleotidesequences between the two DNA molecules (i.e., “regions of homology”).The term “regions of homology” refer to stretches of nucleotide sequenceon nucleic acid fragments that participate in homologous recombinationthat have homology to each other. Effective homologous recombinationwill generally take place where these regions of homology are at leastabout 10 bp in length where at least about 50 bp in length is preferred.Typically fragments that are intended for recombination contain at leasttwo regions of homology where targeted gene disruption or replacement isdesired.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992,111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(hereinafter “Maniatis”); by Silhavy, T. J., Bennan, M. L. and Enquist,L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

Microbial Biosynthesis of Fatty Acids

In general, lipid accumulation in oleaginous microorganisms is triggeredin response to the overall carbon to nitrogen ratio present in thegrowth medium (FIG. 1). When cells have exhausted available nitrogensupplies (e.g., when the carbon to nitrogen ratio is greater than about40), the depletion of cellular adenosine monophosphate (AMP) leads tothe cessation of AMP-dependent isocitrate dehydrogenase activity in themitochondria and the accumulation of citrate, transport of citrate intothe cytosol, and subsequent cleavage of the citrate by ATP-citrate lyaseto yield acetyl-CoA. Acetyl-CoA is the principle building block for denovo biosynthesis of fatty acids. Although any compound that caneffectively be metabolized to produce acetyl-CoA can serve as aprecursor of fatty acids, glucose is the primary source of carbon inthis type of reaction (FIG. 1). Glucose is converted to pyruvate viaglycolysis, and pyruvate is then transported into the mitochondria whereit can be converted to acetyl-CoA by pyruvate dehydrogenase (“PD”).Since acetyl-CoA can not be transported directly across themitochondrial membrane into the cytoplasm, the two carbons fromacetyl-CoA condense with oxaloacetate to yield citrate (catalyzed bycitrate synthase). Citrate is transported directly into the cytoplasm,where it is cleaved by ATP-citrate lyase to regenerate acetyl-CoA andoxaloacetate. The oxaloacetate reenters the tricarboxylic acid cycle,via conversion to malate.

The synthesis of malonyl-CoA is the first committed step of fatty acidbiosynthesis, which takes place in the cytoplasm. Malonyl-CoA isproduced via carboxylation of acetyl-CoA by acetyl-CoA carboxylase(“ACC”). Fatty acid synthesis is catalyzed by a multi-enzyme fatty acidsynthase complex (“FAS”) and occurs by the condensation of eighttwo-carbon fragments (acetyl groups from acetyl-CoA) to form a 16-carbonsaturated fatty acid, palmitate. More specifically, FAS catalyzes aseries of 7 reactions, which involve the following (Smith, S. FASEB J.,8(15):1248-59 (1994)):

-   -   1. Acetyl-CoA and malonyl-CoA are transferred to the acyl        carrier protein (ACP) of FAS. The acetyl group is then        transferred to the malonyl group, forming β-ketobutyryl-ACP and        releasing CO₂.    -   2. The β-ketobutyryl-ACP undergoes reduction (via β-ketoacyl        reductase) and dehydration (via β-hydroxyacyl dehydratase) to        form a trans-monounsaturated fatty acyl group.    -   3. The double bond is reduced by NADPH, yielding a saturated        fatty-acyl group two carbons longer than the initial one. The        butyryl-group's ability to condense with a new malonyl group and        repeat the elongation process is then regenerated.    -   4. When the fatty acyl group becomes 16 carbons long, a        thioesterase activity hydrolyses it, releasing free palmitate.

Palmitate (16:0) is the precursor of longer chain saturated andunsaturated fatty acids (e.g., stearic (18:0), palmitoleic (16:1) andoleic (18:1) acids) through the action of elongases and desaturasespresent in the endoplasmic reticulum membrane. Palmitate and stearate(as CoA and/or ACP esters) are converted to their unsaturatedderivatives, palmitoleic (16:1) and oleic (18:1) acids, respectively, bythe action of a Δ9 desaturase.

Triacylglycerols (the primary storage unit for fatty acids) are formedby the esterification of two molecules of acyl-CoA toglycerol-3-phosphate to yield 1,2-diacylglycerol phosphate (commonlyidentified as phosphatidic acid) (FIG. 1). The phosphate is thenremoved, by phosphatidic acid phosphatase, to yield 1,2-diacylglycerol.Triacylglycerol is formed upon the addition of a third fatty acid, forexample, by the action of a diacylglycerol-acyl transferase.

Biosynthesis of Omega Fatty Acids

Simplistically, the metabolic process that converts LA to GLA, DGLA andARA (the ω-6 pathway) and ALA to STA, ETA, EPA, DPA and DHA (the ω-3pathway) involves elongation of the carbon chain through the addition oftwo-carbon units and desaturation of the molecule through the additionof double bonds (FIG. 2). This requires a series of special desaturationand elongation enzymes present in the endoplasmic reticulum membrane.

ω-6 Fatty Acids

Oleic acid is converted to LA (18:2), the first of the ω-6 fatty acids,by the action of a Δ12 desaturase. Subsequent ω-6 fatty acids areproduced as follows: 1.) LA is converted to GLA by the activity of a Δ6desaturase; 2.) GLA is converted to DGLA by the action of an elongase;and 3.) DGLA is converted to ARA by the action of a Δ5 desaturase.

ω-3 Fatty Acids

Linoleic acid (LA) is converted to ALA, the first of the ω-3 fattyacids, by the action of a Δ15 desaturase. Subsequent ω-3 fatty acids areproduced in a series of steps similar to that for the ω-6 fatty acids.Specifically: 1.) ALA is converted to STA by the activity of a Δ6desaturase; 2.) STA is converted to ETA by the activity of an elongase;and 3.) ETA is converted to EPA by the activity of a Δ5 desaturase.Alternatively, ETA and EPA can be produced from DGLA and ARA,respectively, by the activity of a Δ17 desaturase. EPA can be furtherconverted to DHA by the activity of an elongase and a Δ4 desaturase.

In alternate embodiments, a Δ9 elongase is able to catalyze theconversion of LA and ALA to EDA and ETrA, respectively. A Δ8 desaturasethen converts these products to DGLA and ETA, respectively.

Genes Involved in Omega Fatty Acid Production

Many microorganisms, including algae, bacteria, molds and yeast, cansynthesize PUFAs and omega fatty acids in the ordinary course ofcellular metabolism. Particularly well-studied are fungi includingSchizochytrium aggregatm, species of the genus Thraustochytrium andMorteriella alpina. Additionally, many dinoflagellates (Dinophyceaae)naturally produce high concentrations of PUFAs. As such, a variety ofgenes involved in oil production have been identified through geneticmeans and the DNA sequences of some of these genes are publiclyavailable (non-limiting examples are shown below in Table 3):

TABLE 3 Some Publicly Available Genes involved in PUFA ProductionGenbank Accession No. Description AY131238 Argania spinosa Δ6 desaturaseY055118 Echium pitardii var. pitardii Δ6 desaturase AY055117 Echiumgentianoides Δ6 desaturase AF296076 Mucor rouxii Δ6 desaturase AF007561Borago officinalis Δ6 desaturase L11421 Synechocystis sp. Δ6 desaturaseNM_031344 Rattus norvegicus Δ6 fatty acid desaturase AF465283,Mortierella alpina Δ6 fatty acid desaturase AF465281, AF110510 AF465282Mortierella isabellina Δ6 fatty acid desaturase AF419296 Pythiumirregulare Δ6 fatty acid desaturase AB052086 Mucor circinelloides D6dmRNA for Δ6 fatty acid desaturase AJ250735 Ceratodon purpureus mRNA forΔ6 fatty acid desaturase AF126799 Homo sapiens Δ6 fatty acid desaturaseAF126798 Mus musculus Δ6 fatty acid desaturase AF199596, Homo sapiens Δ5desaturase AF226273 AF320509 Rattus norvegicus liver Δ5 desaturaseAB072976 Mus musculus D5D mRNA for Δ5 desaturase AF489588Thraustochytrium sp. ATCC21685 Δ5 fatty acid desaturase AJ510244Phytophthora megasperma mRNA for Δ5 fatty acid desaturase AF419297Pythium irregulare Δ5 fatty acid desaturase AF07879 Caenorhabditiselegans Δ5 fatty acid desaturase AF067654 Mortierella alpina Δ5 fattyacid desaturase AB022097 Dictyostelium discoideum mRNA for Δ5 fatty aciddesaturase AF489589.1 Thraustochytrium sp. ATCC21685 Δ4 fatty aciddesaturase AX464731 Mortierella alpina elongase gene (also WO 00/12720)AAG36933 Emericella nidulans oleate Δ12 desaturase AF110509, Mortierellaalpina Δ12 fatty acid desaturase mRNA AB020033 AAL13300 Mortierellaalpina Δ12 fatty acid desaturase AF417244 Mortierella alpina ATCC 16266Δ12 fatty acid desaturase gene AF161219 Mucor rouxii Δ12 desaturase mRNAX86736 Spiruline platensis Δ12 desaturase AF240777 Caenorhabditiselegans Δ12 desaturase AB007640 Chlamydomonas reinhardtii Δ12 desaturaseAB075526 Chlorella vulgaris Δ12 desaturase AP002063 Arabidopsis thalianamicrosomal Δ12 desaturase NP_441622, Synechocystis sp. PCC 6803 Δ15desaturase BAA18302, BAA02924 AAL36934 Perilla frutescens Δ15 desaturaseAF338466 Acheta domesticus Δ9 desaturase 3 mRNA AF438199 Picea glaucadesaturase Δ9 (Des9) mRNA E11368 Anabaena Δ9 desaturase E11367Synechocystis Δ9 desaturase D83185 Pichia angusta DNA for Δ9 fatty aciddesaturase U90417 Synechococcus vulcanus Δ9 acyl-lipid fatty aciddesaturase (desC) gene AF085500 Mortierella alpina Δ9 desaturase mRNAAY504633 Emericella nidulans Δ9 stearic acid desaturase (sdeB) geneNM_069854 Caenorhabditis elegans essential fatty acid desaturase,stearoyl-CoA desaturase (39.1 kD) (fat-6) complete mRNA AF230693Brassica oleracea cultivar Rapid Cycling stearoyl-ACP desaturase(Δ9-BO-1) gene, exon sequence AX464731 Mortierella alpina elongase gene(also WO 02/08401) NM_119617 Arabidopsis thaliana fatty acid elongase 1(FAE1) (At4g34520) mRNA NM_134255 Mus musculus ELOVL family member 5,elongation of long chain fatty acids (yeast) (Elovl5), mRNA NM_134383Rattus norvegicus fatty acid elongase 2 (rELO2), mRNA NM_134382 Rattusnorvegicus fatty acid elongase 1 (rELO1), mRNA NM_068396, Caenorhabditiselegans fatty acid ELOngation (elo-6), NM_068392, (elo-5), (elo-2),(elo-3), and (elo-9) mRNA NM_070713, NM_068746, NM_064685

Additionally, the patent literature provides many additional DNAsequences of genes (and/or details concerning several of the genes aboveand their methods of isolation) involved in oil production. See, forexample: U.S. Pat. No. 5,968,809 (Δ6 desaturases); U.S. Pat. Nos.5,972,664 and 6,075,183 (Δ5 desaturases); WO 91/13972 and U.S. Pat. No.5,057,419 (Δ9 desaturases); U.S. 2003/0196217 A1 (Δ17 desaturases); WO02/090493 (Δ4 desaturases); WO 93/11245 and WO 03/099216 (Δ15desaturases); WO 00/12720 and U.S. 2002/0139974A1 (elongases). Each ofthese patents and applications are herein incorporated by reference intheir entirety.

Of particular interest herein are Δ12 desaturases, and morespecifically, Δ12 desaturases that are suitable for heterologousexpression in oleaginous yeast (e.g., Yarrowia lipolytica). Sequences ofsome Δ12 desaturases (i.e., Glycine max, Brassica napus, Arabidopsisthaliana, Ricinus communis, Zea mays; Neurospora crassa, Botrytiscinerea) are disclosed in WO 94/11516 and WO 03/099216.

Additionally, the native Yarrowia lipolytica Δ12 fatty acid desaturasewas recently isolated and characterized (see co-pending U.S. patentapplication Ser. No. 10/840,325, incorporated entirely by reference; seealso Examples 2 and 3 herein and SEQ ID NOs:51 and 52). Briefly, apartial putative Δ12 desaturase DNA fragment from Yarrowia lipolyticawas cloned by PCR using degenerate PCR primers. Targeted disruption ofthe endogenous Yarrowia lipolytica Δ12 desaturase gene using thefragment produced increased levels of 18:1 and no detectable 18:2 in thedisrupted strain, thereby confirming that the native Δ12 desaturaseactivity was eliminated. Subsequently, genomic DNA sequences flankingthe integrated plasmid were isolated using plasmid rescue and afull-length Yarrowia lipolytica Δ12 desaturase gene was assembled (SEQID NO:51). The sequence included an open reading frame of 1257 bases(nucleotides +283 to +1539 of SEQ ID NO:51), while the deduced encodedamino acid sequence was 419 residues in length (SEQ ID NO:52).Over-expression of this Δ12 desaturase was suitable to increase thepercent substrate conversion of oleic acid to LA (calculated as([18:2]/[18:1+18:2])*100), such that it increased from 59% in thewildtype cells to 74% in the transformed host cells. Despite theincreased availability of LA within these host cells, however, it wasdesirable to obtain an even larger substrate pool suitable to enablehigh-level production of a variety of (0-3 and/or ω-6 PUFAs within theY. lipolytica transformant cells. Thus, expression of a heterologousprotein having high-level Δ12 desaturase activity was thereforeadvantageous in the pathway engineering of the organism.

Many factors affect the choice of a specific polypeptide having Δ12desaturase activity that is to be expressed in a host cell forproduction of PUFAs (optionally in combination with other desaturasesand elongases). Depending upon the host cell, the availability ofsubstrate, and the desired end product(s), several polypeptides are ofinterest; however, considerations for choosing a specific polypeptidehaving desaturase activity include the substrate specificity of thepolypeptide, whether the polypeptide or a component thereof is arate-limiting enzyme, whether the desaturase is essential for synthesisof a desired PUFA, and/or co-factors required by the polypeptide. Theexpressed polypeptide preferably has parameters compatible with thebiochemical environment of its location in the host cell. For example,the polypeptide may have to compete for substrate with other enzymes inthe host cell. Analyses of the KM and specific activity of thepolypeptide are therefore considered in determining the suitability of agiven polypeptide for modifying PUFA production in a given host cell.The polypeptide used in a particular host cell is one which can functionunder the biochemical conditions present in the intended host cell butotherwise can be any polypeptide having Δ12 desaturase activity capableof modifying the desired fatty acids (i.e., oleic acid). Thus, thesequences may be derived from any source, e.g., isolated from a naturalsource (from bacteria, algae, fungi, plants, animals, etc.), producedvia a semi-synthetic route or synthesized de novo.

For the purposes of the present invention herein, however, it is mostdesirable for the polypeptide having Δ12 desaturase activity to have aconversion efficiency of at least about 70% when expressed in thedesired host cell, wherein a conversion efficiency of at least about 80%is particularly suitable, and a conversion efficiency of at least about85% is most preferred.

Identification of Novel Fungal Δ12 Desaturases

A novel Δ12 desaturase from Fusarium moniliforme was identified herein,by sequence comparison using the Yarrowia lipolytica Δ12 desaturaseprotein sequence (SEQ ID NO:52) as a query sequence. Specifically, thisYarrowia query sequence was used to search putative encoded proteinsequences of a proprietary DuPont expressed sequence tag (EST) libraryof Fusarium moniliforme strain M-8114 (E.I. du Pont de Nemours and Co.,Inc., Wilmington, Del.). This resulted in the identification of twohomologous sequences, Fm1 (SEQ ID NO:2) and Fm2 (SEQ ID NO:4), encodedby nucleotide sequences SEQ ID NOs:1 and 3, respectively.

The Yarrowia Δ12 desaturase sequence was also used as a query againstpublic databases of several filamentous fungi; specifically, homologousprotein sequences were identified in Aspergillus nidulans (SEQ ID NOs:6and 8), Magnaporthe grisea (SEQ ID NOs:10 and 12), Neurospora crassa(SEQ ID NOs:14 and 16), Fusarium graminearium (SEQ ID NOs:18 and 20),Aspergillus fumigatus (SEQ ID NO:21) and Aspergillus flavus (SEQ IDNO:22). Subsequent phylogenetic and homology analysis, based oncomparison of these sequences (i.e., SEQ ID NOs:2, 4, 6, 8,10, 12, 14,16,18, 20, 21 and 22) using the method of Clustal W (slow, accurate,Gonnet option; Thompson et al. Nucleic Acids Res. 22:46734680 (1994)),revealed two distinct “sub-families” of proteins having homology withthe Yarrowia Δ12 desaturase. Specifically, all proteins of “sub-family1” (SEQ ID NOs:2, 6,10, 14 and 18) were at least 46.2% identical to eachother and were less than 39.6% identical to the proteins of “sub-family2” (SEQ ID NOs:4, 8, 12,16, 20, 21 and 22) (FIGS. 4 and 5; Clustalmethod of alignment (supra)). The proteins of sub-family 2 were at least56.3% identical to each other (see Example 4).

Since Yarrowia is only able to synthesize 18:2 (but not 18:3) while mostof the filamentous fungi described above can make both 18:2 and ALA, andsince Yarrowia has a single Δ12 desaturase while most of the filamentousfungi had two homologs to the Yarrowia Δ12 desaturase, the Applicantspostulated that one of the sub-families of desaturases in theseorganisms represented Δ12 desaturases and the other represented Δ15desaturases. This hypothesis was tested by determining the activity of arepresentative protein within each of the two sub-families usingexpression analysis. Specifically, Fm1 and Mg1 were expressed inYarrowia lipolytica and found to encode Δ15 desaturases (see co-pendingU.S. Provisional Application 60/519,191); similarly, the Dec. 4, 2003publication of WO 03/099216 suggests that the sequences identifiedherein as the sub-family 1 Neurospora crassa and Aspergillus nidulanssequences had Δ15 desaturase activity. In contrast, Fm2 was expressed inY. lipolytica as described herein and was characterized as a Δ12desaturase. The Δ12 desaturase activity of the sub-family 2 Neurosporacrassa sequence was similarly confirmed in WO 03/099216.

The Fusarium moniliforme Δ12 desaturase deduced amino acid sequence (SEQID NO:4) was compared to public database sequences using a Clustalmethod of alignment (Thompson et al., Nucleic Acids Res. 22:46734680(1994)). Thus, the Fusarium moniliforme Δ12 desaturase amino acidsequence was most similar based on percent identity to the Fusariumgraminearium Δ12 desaturase provided herein as SEQ ID NO:20 (95%identical over a length of 477 amino acids). More preferred amino acidfragments are at least about 96% identical to the sequence herein, wherethose sequences that are 97%-98% identical are particularly suitable andthose sequences that are about 99% identical are most preferred.

In like manner, comparison of the Fusarium moniliforme Δ12 desaturasenucleotide base sequence to public databases using the Clustal method ofalignment reveals that the most similar known nucleic acid sequence(Contig 1.233 in the F. graminearium genome project; SEQ ID NO:19herein) is about 89.2% identical to the nucleic acid sequence of theFusarium moniliforme Δ12 desaturase reported herein (SEQ ID NO:3).Preferred Δ12 desaturase encoding nucleic acid sequences correspondingto the instant ORF are those encoding active proteins and which are atleast about 89%-90% identical to the nucleic acid sequence encoding theFusarium moniliforme Δ12 desaturase reported herein, where thosesequences that are 91%-95% identical are particularly suitable and thosesequences that are greater than 95% identical are most preferred.

Identification and Isolation of Homologs

The Δ12 desaturase nucleic acid fragment of the instant invention may beused to identify and isolate genes encoding homologous proteins from thesame or other bacterial, algal, fungal or plant species.

Identification Techniques

For example, a substantial portion of the Fusarium moniliforme Δ12desaturase amino acid or nucleotide sequence described herein can beused to putatively identify related polypeptides or genes, either bymanual evaluation of the sequence by one skilled in the art, or bycomputer-automated sequence comparison and identification usingalgorithms such as BLAST (Basic Local Alignment Search Tool; Altschul,S. F., et al., J. Mol. Biol. 215:403-410 (1993)) and ClustalW (Megalignprogram of DNASTAR software). As described above, use of the Yarrowialipolytica Δ12 desaturase (SEQ ID NO:52) permitted the identification ofa suite of fungal desaturases which, upon analysis, clustered as twodistinct sub-families of proteins (i.e., sub-family 1 and sub-family 2).Subfamily-2 comprised the Fusarium moniliforme Δ12 desaturase describedabove, as well as the proteins whose coding DNA sequences are foundwithin the following:

-   -   Contig 1.15 (scaffold 1) (AAG36933) in the Aspergillus nidulans        genome project (sponsored by the Center for Genome Research        (CGR), Cambridge, Mass.) (SEQ ID NO:8);    -   Locus MG01985.1 in contig 2.375 in the Magnaporthe grisea genome        project (sponsored by the CGR and International Rice Blast        Genome Consortium) (SEQ ID NO:12);    -   GenBank Accession No. MBX01000374 (Neurospora crassa) (SEQ ID        NO:16);    -   Contig 1.233 in the Fusarium graminearium genome project        (sponsored by the CGR and the International Gibberella zeae        Genomics Consortium (IGGR)) (SEQ ID NO:20);    -   AFA.344248:345586 (reverse) in the Aspergillus fumigatus genome        project (sponsored by Sanger Institute, collaborators at the        University of Manchester and The Institute of Genome Research        (TIGR)) (SEQ ID NO:21); and,    -   GenBank Accession No. AY280867 (Aspergillus flavus) (SEQ ID        NO:22).        Each of the above proteins are hypothesized to encode a Δ12        desaturase. This hypothesis was confirmed for Neurospora crassa        in WO 03/099216.

Analysis of the above proteins revealed that these proteins have atleast 56.3% sequence identity to the Fusarium moniliforme Δ12 desaturase(SEQ ID NO:4), according to the Clustal method of alignment (supra)(FIG. 5). Additionally, the Δ12 desaturases of sub-family 2 in thepresent invention were also compared to other known Δ12 desaturaseproteins; however, the Δ12 desaturases of sub-family 2 herein are morehomologous to the Yarrowia lipolytica Δ12 desaturase (51.6% identity;FIG. 6) than they are to any other known Δ12 desaturase. One skilled inthe art would be able to use similar methodology to identify otherorthologous proteins that would also cluster within sub-family 2(identified herein as Δ12 desaturases).

Alternatively, any of the instant desaturase sequences (i.e., SEQ IDNOs:3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 21 and 22) may be employed ashybridization reagents for the identification of homologs. The basiccomponents of a nucleic acid hybridization test include a probe, asample suspected of containing the gene or gene fragment of interest anda specific hybridization method. Probes of the present invention aretypically single-stranded nucleic acid sequences that are complementaryto the nucleic acid sequences to be detected. Probes are “hybridizable”to the nucleic acid sequence to be detected. The probe length can varyfrom 5 bases to tens of thousands of bases, and will depend upon thespecific test to be done. Typically a probe length of about 15 bases toabout 30 bases is suitable. Only part of the probe molecule need becomplementary to the nucleic acid sequence to be detected. In addition,the complementarity between the probe and the target sequence need notbe perfect. Hybridization does occur between imperfectly complementarymolecules with the result that a certain fraction of the bases in thehybridized region are not paired with the proper complementary base.

Hybridization methods are well defined. Typically the probe and samplemust be mixed under conditions that will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed. Optionally, achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature (Van Ness and Chen, Nucl. Acids Res.19:5143-5151 (1991)). Suitable chaotropic agents include guanidiniumchloride, guanidinium thiocyanate, sodium thiocyanate, lithiumtetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate,potassium iodide, and cesium trifluoroacetate, among others. Typically,the chaotropic agent will be present at a final concentration of about 3M. If desired, one can add formamide to the hybridization mixture,typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 Mbuffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), orbetween 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal),polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g.,calf thymus or salmon sperm DNA, or yeast RNA), and optionally fromabout 0.5 to 2% wt/vol glycine. Other additives may also be included,such as volume exclusion agents that include a variety of polarwater-soluble or swellable agents (e.g., polyethylene glycol), anionicpolymers (e.g., polyacrylate or polymethylacrylate) and anionicsaccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit immobilized nucleic acid probe that is unlabeled and complementary toone portion of the sequence.

Isolation Methods

The Fusarium moniliforme Δ12 desaturase nucleic acid fragment of theinstant invention (or any of the Δ12 desaturases identified herein [SEQID NOs:7, 8, 11, 12, 15, 16, and 19-22]) may be used to isolate genesencoding homologous proteins from the same or other bacterial, algal,fungal or plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art.

Examples of sequence-dependent protocols include, but are not limitedto: 1.) methods of nucleic acid hybridization; 2.) methods of DNA andRNA amplification, as exemplified by various uses of nucleic acidamplification technologies [e.g., polymerase chain reaction (PCR),Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR),Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or stranddisplacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci.U.S.A., 89:392 (1992)]; and 3.) methods of library construction andscreening by complementation.

For example, genes encoding similar proteins or polypeptides to thedesaturases described herein could be isolated directly by using all ora portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired yeast or fungus usingmethodology well known to those skilled in the art (wherein those yeastor fungus producing LA [or LA-derivatives] would be preferred). Specificoligonucleotide probes based upon the instant nucleic acid sequences canbe designed and synthesized by methods known in the art (Maniatis,supra). Moreover, the entire sequences can be used directly tosynthesize DNA probes by methods known to the skilled artisan (e.g.,random primers DNA labeling, nick translation or end-labelingtechniques), or RNA probes using available in vitro transcriptionsystems. In addition, specific primers can be designed and used toamplify a part of (or full-length of) the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full-length DNA fragments under conditions ofappropriate stringency.

Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art (Thein and Wallace, “The use of oligonucleotide asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp33-50, IRL: Herndon, V A; and Rychlik, W., In Methods in MolecularBiology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols:Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the instant sequences may be used inpolymerase chain reaction protocols to amplify longer nucleic acidfragments encoding homologous genes from DNA or RNA. The polymerasechain reaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from the instantnucleic acid fragments, and the sequence of the other primer takesadvantage of the presence of the polyadenylic acid tracts to the 3′ endof the mRNA precursor encoding microbial genes.

Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) togenerate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (Gibco/BRL,Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated(Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217(1989)).

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of DNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequence may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen DNA expression libraries toisolate full-length DNA clones of interest (Lerner, R. A. Adv. Immunol.36:1 (1984); Maniatis, supra).

Gene Optimization for Improved Heterologous Expression

A variety of techniques can be utilized to improve expression of aparticular Δ12 desaturase of interest in an alternative host. Two suchtechniques include codon-optimization and mutagenesis of the gene.

Codon Optimization

For some embodiments, it may be desirable to modify a portion of thecodons encoding polypeptides having Δ12 desaturase activity, forexample, to enhance the expression of the genes encoding thosepolypeptides in an alternative host (i.e., oleaginous yeast).

In general, host preferred codons can be determined within a particularhost species of interest by examining codon usage in proteins(preferably those proteins expressed in the largest amount) anddetermining which codons are used with highest frequency. Then, thecoding sequence for the polypeptide of interest having desaturaseactivity can be synthesized in whole or in part using the codonspreferred in the host species. All (or portions) of the DNA also can besynthesized to remove any destabilizing sequences or regions ofsecondary structure that would be present in the transcribed mRNA. All(or portions) of the DNA also can be synthesized to alter the basecomposition to one more preferable in the desired host cell.

In preferred embodiments of the invention, the Δ12 desaturases frome.g., Fusarium moniliforme, Aspergillus nidulans, Magnaporthe grisea,Neurospora crassa, Fusarium graminearium, Aspergillus fumigatus andAspergillus flavus could be codon-optimized prior to their expression ina heterologous oleaginous yeast host, such as Yarrowia lipolytica.

Mutagenesis

Methods for synthesizing sequences and bringing sequences together arewell established in the literature. For example, in vitro mutagenesisand selection, site-directed mutagenesis, error prone PCR (Melnikov etal., Nucleic Acids Research, 27(4):1056-1062 (Feb. 15, 20 1999)), “geneshuffling” (U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; and5,837,458) or other means can be employed to obtain mutations ofnaturally occurring desaturase genes, such as the Δ12 desaturasesdescribed herein. This would permit production of a polypeptide havingdesaturase activity in vivo with more desirable physical and kineticparameters for function in the host cell (e.g., a longer half-life or ahigher rate of production of a desired PUFA).

If desired, the regions of a desaturase polypeptide important forenzymatic activity can be determined through routine mutagenesis,expression of the resulting mutant polypeptides and determination oftheir activities. Mutants may include deletions, insertions and pointmutations, or combinations thereof. A typical functional analysis beginswith deletion mutagenesis to determine the N- and C-terminal limits ofthe protein necessary for function, and then internal deletions,insertions or point mutants are made to further determine regionsnecessary for function. Other techniques such as cassette mutagenesis ortotal synthesis also can be used. Deletion mutagenesis is accomplished,for example, by using exonucleases to sequentially remove the 5′ or 3′coding regions. Kits are available for such techniques. After deletion,the coding region is completed by ligating oligonucleotides containingstart or stop codons to the deleted coding region after the 5′ or 3′deletion, respectively. Alternatively, oligonucleotides encoding startor stop codons are inserted into the coding region by a variety ofmethods including site-directed mutagenesis, mutagenic PCR or byligation onto DNA digested at existing restriction sites. Internaldeletions can similarly be made through a variety of methods includingthe use of existing restriction sites in the DNA, by use of mutagenicprimers via site-directed mutagenesis or mutagenic PCR. Insertions aremade through methods such as linker-scanning mutagenesis, site-directedmutagenesis or mutagenic PCR. Point mutations are made throughtechniques such as site-directed mutagenesis or mutagenic PCR.

Chemical mutagenesis also can be used for identifying regions of adesaturase polypeptide important for activity. A mutated construct isexpressed, and the ability of the resulting altered protein to functionas a desaturase is assayed. Such structure-function analysis candetermine which regions may be deleted, which regions tolerateinsertions, and which point mutations allow the mutant protein tofunction in substantially the same way as the native desaturase. Allsuch mutant proteins and nucleotide sequences encoding them that arederived from the desaturase genes described herein are within the scopeof the present invention.

Thus, the present invention comprises the complete sequences of the Δ12desaturase genes as reported in the accompanying Sequence Listing, thecomplement of those complete sequences, substantial portions of thosesequences, codon-optimized desaturases derived therefrom, and thosesequences that are substantially homologous thereto.

Microbial Production Of ω-3 and/or ω-6 Fatty Acids

Microbial production of ω-3 and/or ω-6 fatty acids can have severaladvantages over purification from natural sources such as fish orplants. For example:

-   -   1.) Many microbes are known with greatly simplified oil        compositions compared with those of higher organisms, making        purification of desired components easier;    -   2.) Microbial production is not subject to fluctuations caused        by external variables, such as weather and food supply;    -   3.) Microbially produced oil is substantially free of        contamination by environmental pollutants;    -   4.) Microbes can provide PUFAs in particular forms which may        have specific uses; and    -   5.) Microbial oil production can be manipulated by controlling        culture conditions, notably by providing particular substrates        for microbially expressed enzymes, or by addition of compounds        or genetic engineering approaches to suppress undesired        biochemical pathways.        In addition to these advantages, production of ω-3 and/or ω-6        fatty acids from recombinant microbes provides the ability to        alter the naturally occurring microbial fatty acid profile by        providing new biosynthetic pathways in the host or by        suppressing undesired pathways, thereby increasing levels of        desired PUFAs (or conjugated forms thereof) and decreasing        levels of undesired PUFAs (see co-pending U.S. patent        application Ser. No. 10/840,579, herein incorporated entirely by        reference).

Methods for Production of Various ω-3 and/or ω-6 Fatty Acids

It is expected that introduction of chimeric genes encoding the Δ12desaturases described herein, under the control of the appropriatepromoters will result in increased production of LA in the transformedhost organism. As such, the present invention encompasses a method forthe direct production of PUFAs comprising exposing a fatty acidsubstrate (i.e., oleic acid) to the PUFA enzyme(s) described herein(e.g., the Fusarium moniliforme Δ12 desaturase), such that the substrateis converted to the desired fatty acid product (i.e., LA). Morespecifically, it is an object of the present invention to provide amethod for the production of LA in an oleaginous yeast, wherein theoleaginous yeast is provided: (a) an isolated nucleic acid fragmentencoding a fungal protein having Δ12 desaturase activity that has atleast 56.3% identity based on the Clustal method of alignment whencompared to a polypeptide having the sequence as set forth in SEQ IDNO:4; and, (b) a source of desaturase substrate consisting of oleicacid; wherein the yeast is grown under conditions such that the chimericdesaturase gene is expressed and the oleic acid is converted to LA, andwherein the LA is optionally recovered. Thus, this method minimallyincludes the use of the following Δ12 desaturases: SEQ ID NOs:4, 8, 12,16, 20, 21 and 22, as described herein.

Alternatively, each PUFA gene and its corresponding enzyme productdescribed herein can be used indirectly for the production of ω-3 and/orω-6 PUFAs. Indirect production of PUFAs occurs wherein the fatty acidsubstrate is converted indirectly into the desired fatty acid product,via means of an intermediate step(s) or pathway intermediate(s). Thus,it is contemplated that the Δ12 desaturases described herein may beexpressed in conjunction with one or more genes that encode otherenzymes, such that a series of reactions occur to produce a desiredproduct. In a preferred embodiment, for example, a host organism may beco-transformed with a vector comprising additional genes encodingenzymes of the PUFA biosynthetic pathway to result in higher levels ofproduction of ω-3 and/or ω-6 fatty acids (e.g., GLA, DGLA, ARA, ALA,STA, ETA, EPA, DPA and DHA). Specifically, for example, it may bedesirable to over-express any one of the Δ12 desaturases describedherein in host cells that are also expressing: 1.) a gene encoding a Δ6desaturase for the overproduction of GLA; 2.) an expression cassettecomprising genes encoding a Δ6 desaturase and a high-affinity elongasefor the overproduction of DGLA; 3.) genes encoding a Δ6 desaturase,high-affinity elongase and Δ5 desaturase for the overproduction of ARA;or 4.) genes encoding a Δ6 desaturase, high-affinity elongase, Δ5desaturase and Δ17 desaturase for the overproduction of EPA. Inalternative embodiments, for example, it may be desirable to overexpressthe Δ12 desaturase as described herein in cells that are alsoexpressing: 1.) a gene encoding a Δ15 desaturase for the overproductionof ALA; 2.) genes encoding a Δ15 desaturase and Δ6 desaturase for theoverproduction of STA; 3.) genes encoding a Δ15 desaturase, Δ6desaturase and a high-affinity elongase for the overproduction of ETA;or 4.) genes encoding a Δ15 desaturase, Δ6 desaturase, high-affinityelongase and Δ5 desaturase for the overproduction of EPA. As is wellknown to one skilled in the art, various other combinations of thefollowing enzymatic activities may be useful to express in a host inconjunction with the desaturase(s) herein: a Δ15 desaturase, a Δ4desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ17 desaturase, a Δ9desaturase, a Δ8 desaturase and/or an elongase(s) (see FIG. 2). Theparticular genes included within a particular expression cassette willdepend on the host cell (and its PUFA profile and/or desaturaseprofile), the availability of substrate, and the desired end product(s).

In alternative embodiments, it may be useful to disrupt a hostorganism's native Δ12 desaturase, based on the complete sequencesdescribed herein, the complement of those complete sequences,substantial portions of those sequences, codon-optimized desaturasesderived therefrom and those sequences that are substantially homologousthereto. For example, the targeted disruption of the Δ12 desaturase in ahost organism produces a mutant strain that is unable to synthesize LA.

Expression Systems, Cassettes And Vectors

The gene and gene product of the instant sequences described herein maybe produced in heterologous microbial host cells, particularly in thecells of oleaginous yeast (e.g., Yarrowia lipolytica). Expression inrecombinant microbial hosts may be useful for the production of variousPUFA pathway intermediates, or for the modulation of PUFA pathwaysalready existing in the host for the synthesis of new productsheretofore not possible using the host.

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of any of the geneproducts of the instant sequences. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh-level expression of the encoded enzymes.

Vectors or DNA cassettes useful for the transformation of suitable hostcells are well known in the art. The specific choice of sequencespresent in the construct is dependent upon the desired expressionproducts (supra), the nature of the host cell and the proposed means ofseparating transformed cells versus non-transformed cells. Typically,however, the vector or cassette contains sequences directingtranscription and translation of the relevant gene(s), a selectablemarker and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene thatcontrols transcriptional initiation and a region 3′ of the DNA fragmentthat controls transcriptional termination. It is most preferred whenboth control regions are derived from genes from the transformed hostcell, although it is to be understood that such control regions need notbe derived from the genes native to the specific species chosen as aproduction host.

Initiation control regions or promoters which are useful to driveexpression of the instant ORFs in the desired host cell are numerous andfamiliar to those skilled in the art. Virtually any promoter capable ofdirecting expression of these genes in the selected host cell issuitable for the present invention. Expression in a host cell can beaccomplished in a transient or stable fashion. Transient expression canbe accomplished by inducing the activity of a regulatable promoteroperably linked to the gene of interest. Stable expression can beachieved by the use of a constitutive promoter operably linked to thegene of interest. As an example, when the host cell is yeast,transcriptional and translational regions functional in yeast cells areprovided, particularly from the host species. The transcriptionalinitiation regulatory regions can be obtained, for example, from: 1.)genes in the glycolytic pathway, such as alcohol dehydrogenase,glyceraldehyde-3-phosphate-dehydrogenase (see U.S. patent applicationSer. No. 10/869,630), phosphoglycerate mutase (see U.S. patentapplication Ser. No. 10/869,630), fructose-bisphosphate aldolase (seeU.S. Patent Application No. 60/519,971), phosphoglucose-isomerase,phosphoglycerate kinase, glycerol-3-phosphate O-acyltransferase (seeU.S. Patent Application No. 60/610,060), etc.; or, 2.) regulatable genessuch as acid phosphatase, lactase, metallothionein, glucoamylase, thetranslation elongation factor EF1-α (TEF) protein (U.S. Pat. No.6,265,185), ribosomal protein S7 (U.S. Pat. No. 6,265,185), etc. Any oneof a number of regulatory sequences can be used, depending upon whetherconstitutive or induced transcription is desired, the efficiency of thepromoter in expressing the ORF of interest, the ease of construction andthe like.

Nucleotide sequences surrounding the translational initiation codon ATGhave been found to affect expression in yeast cells. If any of theinstant Δ12 desaturases are poorly expressed in yeast, the nucleotidesequences of exogenous genes can be modified to include an efficientyeast translation initiation sequence to obtain optimal gene expression.For expression in yeast, this can be done by site-directed mutagenesisof an inefficiently expressed gene by fusing it in-frame to anendogenous yeast gene, preferably a highly expressed gene.Alternatively, one can determine the consensus translation initiationsequence in the host and engineer this sequence into heterologous genesfor their optimal expression in the host of interest (see, e.g., U.S.patent application Ser. No. 10/840,478 for specific teachings applicablefor Yarrowia lipolytica).

The termination region can be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known and functionsatisfactorily in a variety of hosts (when utilized both in the same anddifferent genera and species from where they were derived). Thetermination region usually is selected more as a matter of conveniencerather than because of any particular property. Preferably, thetermination region is derived from a yeast gene, particularlySaccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces.The 3′-regions of mammalian genes encoding γ-interferon and α-2interferon are also known to function in yeast. Termination controlregions may also be derived from various genes native to the preferredhosts. Optionally, a termination site may be unnecessary; however, it ismost preferred if included.

As one of skill in the art is aware, merely inserting a gene into acloning vector does not ensure that it will be successfully expressed atthe level needed. In response to the need for a high expression rate,many specialized expression vectors have been created by manipulating anumber of different genetic elements that control aspects oftranscription, translation, protein stability, oxygen limitation, andsecretion from the host cell. More specifically, some of the molecularfeatures that have been manipulated to control gene expression include:1.) the nature of the relevant transcriptional promoter and terminatorsequences; 2.) the number of copies of the cloned gene and whether thegene is plasmid-borne or integrated into the genome of the host cell;3.) the final cellular location of the synthesized foreign protein; 4.)the efficiency of translation in the host organism; 5.) the intrinsicstability of the cloned gene protein within the host cell; and 6.) thecodon usage within the cloned gene, such that its frequency approachesthe frequency of preferred codon usage of the host cell. Each of thesetypes of modifications are encompassed in the present invention, asmeans to further optimize expression of the Δ12 desaturases describedherein.

Transformation of Microbial Hosts

Once the DNA encoding a polypeptide suitable for expression in anoleaginous yeast has been obtained, it is placed in a plasmid vectorcapable of autonomous replication in a host cell, or it is directlyintegrated into the genome of the host cell. Integration of expressioncassettes can occur randomly within the host genome or can be targetedthrough the use of constructs containing regions of homology with thehost genome sufficient to target recombination with the host locus.Where constructs are targeted to an endogenous locus, all or some of thetranscriptional and translational regulatory regions can be provided bythe endogenous locus.

Where two or more genes are expressed from separate replicating vectors,it is desirable that each vector has a different means of selection andshould lack homology to the other construct(s) to maintain stableexpression and prevent reassortment of elements among constructs.Judicious choice of regulatory regions, selection means and method ofpropagation of the introduced construct(s) can be experimentallydetermined so that all introduced genes are expressed at the necessarylevels to provide for synthesis of the desired products.

Constructs comprising the gene of interest may be introduced into a hostcell by any standard technique. These techniques include transformation(e.g., lithium acetate transformation [Methods in Enzymology,194:186-187 (1991)]), protoplast fusion, biolistic impact,electroporation, microinjection, or any other method that introduces thegene of interest into the host cell. More specific teachings applicablefor oleaginous yeast (i.e., Yarrowia lipolytica) include U.S. Pat. Nos.4,880,741 and 5,071,764 and Chen, D. C. et al. (Appl MicrobiolBiotechnol. 48(2):232-235-(1997)).

For convenience, a host cell that has been manipulated by any method totake up a DNA sequence (e.g., an expression cassette) will be referredto as “transformed” or “recombinant” herein. The transformed host willhave at least one copy of the expression construct and may have two ormore, depending upon whether the gene is integrated into the genome,amplified, or is present on an extrachromosomal element having multiplecopy numbers. The transformed host cell can be identified by selectionfor a marker contained on the introduced construct. Alternatively, aseparate marker construct may be co-transformed with the desiredconstruct, as many transformation techniques introduce many DNAmolecules into host cells. Typically, transformed hosts are selected fortheir ability to grow on selective media. Selective media mayincorporate an antibiotic or lack a factor necessary for growth of theuntransformed host, such as a nutrient or growth factor. An introducedmarker gene may confer antibiotic resistance, or encode an essentialgrowth factor or enzyme, thereby permitting growth on selective mediawhen expressed in the transformed host. Selection of a transformed hostcan also occur when the expressed marker protein can be detected, eitherdirectly or indirectly. The marker protein may be expressed alone or asa fusion to another protein. The marker protein can be detected by: 1.)its enzymatic activity (e.g., β-galactosidase can convert the substrateX-gal [5-bromo4-chloro-3-indolyl-β-D-galactopyranoside] to a coloredproduct; luciferase can convert luciferin to a light-emitting product);or 2.) its light-producing or modifying characteristics (e.g., the greenfluorescent protein of Aequorea victoria fluoresces when illuminatedwith blue light). Alternatively, antibodies can be used to detect themarker protein or a molecular tag on, for example, a protein ofinterest. Cells expressing the marker protein or tag can be selected,for example, visually, or by techniques such as FACS or panning usingantibodies. For selection of yeast transformants, any marker thatfunctions in yeast may be used. Desirably, resistance to kanamycin,hygromycin and the amino glycoside G418 are of interest, as well asability to grow on media lacking uracil or leucine.

Following transformation, substrates suitable for the instant Δ12desaturases (and, optionally other PUFA enzymes that are co-expressedwithin the host cell) may be produced by the host either naturally ortransgenically, or they may be provided exogenously.

Metabolic Engineering of ω-3 and/or ω-6 Fatty Acid Biosynthesis inMicrobes

Knowledge of the sequences of the present Δ12 desaturases will be usefulfor manipulating ω-3 and/or ω-6 fatty acid biosynthesis in oleaginousyeast, and particularly, in Yarrowia lipolytica. This may requiremetabolic engineering directly within the PUFA biosynthetic pathway oradditional manipulation of pathways that contribute carbon to the PUFAbiosynthetic pathway. Methods useful for manipulating biochemicalpathways are well known to those skilled in the art.

Techniques to Up-Requlate Desirable Biosynthetic Pathways

Additional copies of desaturase (and optionally elongase) genes may beintroduced into the host to increase the output of ω-3 and/or ω-6 fattyacid biosynthesis pathways, typically through the use of multicopyplasmids. Expression of desaturase and elongase genes also can beincreased at the transcriptional level through the use of a strongerpromoter (either regulated or constitutive) to cause increasedexpression, by removing/deleting destabilizing sequences from either themRNA or the encoded protein, or by adding stabilizing sequences to themRNA (U.S. Pat. No. 4,910,141). Yet another approach to increaseexpression of heterologous desaturase or elongase genes is to increasethe translational efficiency of the encoded mRNAs by replacement ofcodons in the native gene with those for optimal gene expression in theselected host microorganism.

Techniques to Down-Regulate Undesirable Biosynthetic Pathways

Conversely, biochemical pathways competing with the ω-3 and/or ω-6 fattyacid biosynthesis pathways for energy or carbon, or native PUFAbiosynthetic pathway enzymes that interfere with production of aparticular PUFA end-product, may be eliminated by gene disruption ordown-regulated by other means (e.g., antisense mRNA). For genedisruption, a foreign DNA fragment (typically a selectable marker gene)is inserted into the structural gene to be disrupted in order tointerrupt its coding sequence and thereby functionally inactivate thegene. Transformation of the disruption cassette into the host cellresults in replacement of the functional native gene by homologousrecombination with the non-functional disrupted gene (See, for example:Hamilton et al. J. Bacteriol. 171:4617-4622 (1989); Balbas et al. Gene136:211-213 (1993); Gueldener et al. Nucleic Acids Res. 24:2519-2524(1996); and Smith et al. Methods Mol. Cell. Biol. 5:270-277(1996)).

Antisense technology is another method of down-regulating genes when thesequence of the target gene is known. To accomplish this, a nucleic acidsegment from the desired gene is cloned and operably linked to apromoter such that the anti-sense strand of RNA will be transcribed.This construct is then introduced into the host cell and the antisensestrand of RNA is produced. Antisense RNA inhibits gene expression bypreventing the accumulation of mRNA that encodes the protein ofinterest. The person skilled in the art will know that specialconsiderations are associated with the use of antisense technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of antisense genes may require the use of differentchimeric genes utilizing different regulatory elements known to theskilled artisan.

Although targeted gene disruption and antisense technology offereffective means of down-regulating genes where the sequence is known,other less specific methodologies have been developed that are notsequence-based. For example, cells may be exposed to UV radiation andthen screened for the desired phenotype. Mutagenesis with chemicalagents is also effective for generating mutants and commonly usedsubstances include chemicals that affect nonreplicating DNA (e.g., HNO₂and NH₂OH), as well as agents that affect replicating DNA (e.g.,acridine dyes, notable for causing frameshift mutations). Specificmethods for creating mutants using radiation or chemical agents are welldocumented in the art. See, for example: Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, 2^(nd) ed. (1989)Sinauer Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl.Biochem. Biotechnol., 36: 227 (1992).

Another non-specific method of gene disruption is the use oftransposable elements or transposons. Transposons are genetic elementsthat insert randomly into DNA but can be later retrieved on the basis ofsequence to determine where the insertion has occurred. Both in vivo andin vitro transposition methods are known. Both methods involve the useof a transposable element in combination with a transposase enzyme. Whenthe transposable element or transposon is contacted with a nucleic acidfragment in the presence of the transposase, the transposable elementwill randomly insert into the nucleic acid fragment. The technique isuseful for random mutagenesis and for gene isolation, since thedisrupted gene may be identified on the basis of the sequence of thetransposable element. Kits for in vitro transposition are commerciallyavailable [see, for example: 1.) The Primer Island Transposition Kit,available from Perkin Elmer Applied Biosystems, Branchburg, N.J., basedupon the yeast Ty1 element; 2.) The Genome Priming System, availablefrom New England Biolabs, Beverly, Mass., based upon the bacterialtransposon Tn7; and 3.) the EZ::TN Transposon Insertion Systems,available from Epicentre Technologies, Madison, Wis., based upon the Tn5bacterial transposable element].

Within the context of the present invention, it may be useful tomodulate the expression of the fatty acid biosynthetic pathway by anyone of the methods described above. For example, the present inventionprovides genes (i.e., Δ12 desaturases) encoding key enzymes in thebiosynthetic pathways leading to the production of ω-3 and/or ω-6 fattyacids. It will be particularly useful to express these genes inoleaginous yeast that produce insufficient amounts of 18:2 fatty acidsand to modulate the expression of this and other PUFA biosynthetic genesto maximize production of preferred PUFA products using various meansfor metabolic engineering of the host organism. Likewise, to maximizePUFA production with these genes, it may be necessary to disruptpathways that compete for the carbon flux directed toward PUFAbiosynthesis.

In alternate embodiments, it may be desirable to disrupt the Δ12desaturase herein, to prevent synthesis of ω-3 and/or ω-6 fatty acids.In another alternate embodiment it will be possible to regulate theproduction of ω-3 and/or ω-6 fatty acids by placing any of the presentΔ12 desaturase genes under the control of an inducible or regulatedpromoter.

Preferred Microbial Hosts for Recombinant Expression of Δ12 Desaturases

Host cells for expression of the instant genes and nucleic acidfragments may include microbial hosts that grow on a variety offeedstocks, including simple or complex carbohydrates, organic acids andalcohols, and/or hydrocarbons over a wide range of temperature and pHvalues. Although the genes described in the instant invention have beenisolated for expression in an oleaginous yeast, and in particularYarrowia lipolytica, it is contemplated that because transcription,translation and the protein biosynthetic apparatus is highly conserved,any bacteria, yeast, algae and/or filamentous fungus will be a suitablehost for expression of the present nucleic acid fragments.

Preferred hosts are oleaginous organisms, such as oleaginous yeast.These oleaginous organisms are naturally capable of oil synthesis andaccumulation, wherein the oil can comprise greater than about 25% of thecellular dry weight, more preferably greater than about 30% of thecellular dry weight, and most preferably greater than about 40% of thecellular dry weight. Genera typically identified as oleaginous yeastinclude, but are not limited to: Yarrowia, Candida, Rhodotorula,Rhodospordium, Cryptococcus, Trichosporon and Lipomyces. Morespecifically, illustrative oil-synthesizing yeast include:Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candidarevkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporonpullans, T. cutaneum, Rhodotorula glutinus, R. graminis and Yarrowialipolytica (formerly classified as Candida lipolytica).

Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in afurther embodiment, most preferred are the Yarrowia lipolytica strainsdesignated as ATCC #76982, ATCC #20362, ATCC #8862, ATCC #18944 and/orLGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol.82(1):43-9 (2002)).

Other preferred microbial hosts include oleaginous bacteria, algae andother fungi (e.g., Thraustochytrium sp., Schizochytrium sp. andMortierella sp.).

Fermentation Processes for PUFA Production

The transformed microbial host cell is grown under conditions thatoptimize activity of fatty acid biosynthetic genes and produce thegreatest and the most economical yield of fatty acids (e.g., LA, whichcan in turn increase the production of various ω-3 and/or ω-6 fattyacids). In general, media conditions that may be optimized include thetype and amount of carbon source, the type and amount of nitrogensource, the carbon-to-nitrogen ratio, the oxygen level, growthtemperature, pH, length of the biomass production phase, length of theoil accumulation phase and the time of cell harvest. Microorganisms ofinterest, such as oleaginous yeast, are grown in complex media (e.g.,yeast extract-peptone-dextrose broth (YPD)) or a defined minimal mediathat lacks a component necessary for growth and thereby forces selectionof the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.)).

Fermentation media in the present invention must contain a suitablecarbon source. Suitable carbon sources may include, but are not limitedto: monosaccharides (e.g., glucose, fructose), disaccharides (e.g.,lactose or sucrose), oligosaccharides, polysaccharides (e.g., starch,cellulose or mixtures thereof), sugar alcohols (e.g., glycerol) ormixtures from renewable feedstocks (e.g., cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt). Additionally,carbon sources may include alkanes, fatty acids, esters of fatty acids,monoglycerides, diglycerides, triglycerides, phospholipids and variouscommercial sources of fatty acids including vegatable oils (e.g.,soybean oil) and animal fats. Additionally, the carbon substrate mayinclude one-carbon substrates (e.g., carbon dioxide or methanol) forwhich metabolic conversion into key biochemical intermediates has beendemonstrated. Hence it is contemplated that the source of carbonutilized in the present invention may encompass a wide variety ofcarbon-containing substrates and will only be limited by the choice ofthe host organism. Although all of the above mentioned carbon substratesand mixtures thereof are expected to be suitable in the presentinvention, preferred carbon substrates are sugars and/or fatty acids.Most preferred is glucose and/or fatty acids containing between 10-22carbons.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organicsource (e.g., urea or glutamate). In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins, and other componentsknown to those skilled in the art, suitable for the growth of themicroorganism and promotion of the enzymatic pathways necessary for PUFAproduction. Particular attention is given to several metal ions (e.g.,Mn⁺², Co⁺², Zn⁺², Mg⁺²).that promote synthesis of lipids and PUFAs(Nakahara, T. et al. Ind. Appl. Single Cell Oils, D. J. Kyle and R.Colin, eds. pp 61-97 (1992)).

Preferred growth media in the present invention are common commerciallyprepared media, such as Yeast Nitrogen Base (DIFCO Laboratories,Detroit, Mich.). Other defined or synthetic growth media may also beused and the appropriate medium for growth of the particularmicroorganism will be known by one skilled in the art of microbiology orfermentation science. A suitable pH range for the fermentation istypically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 ispreferred as the range for the initial growth conditions. Thefermentation may be conducted under aerobic or anaerobic conditions,wherein microaerobic conditions are preferred.

Typically, accumulation of high levels of PUFAs in oleaginous yeastcells requires a two-stage process, since the metabolic state must be“balanced” between growth and synthesis/storage of fats. Thus, mostpreferably, a two-stage fermentation process is necessary for theproduction of PUFAs in oleaginous yeast. In this approach, the firststage of the fermentation is dedicated to the generation andaccumulation of cell mass and is characterized by rapid cell growth andcell division. In the second stage of the fermentation, it is preferableto establish conditions of nitrogen deprivation in the culture topromote high levels of lipid accumulation. The effect of this nitrogendeprivation is to reduce the effective concentration of AMP in thecells, thereby reducing the activity of the NAD-dependent isocitratedehydrogenase of mitochondria. When this occurs, citric acid willaccumulate, thus forming abundant pools of acetyl-CoA in the cytoplasmand priming fatty acid synthesis. Thus, this phase is characterized bythe cessation of cell division followed by the synthesis of fatty acidsand accumulation of oil.

Although cells are typically grown at about 30° C., some studies haveshown increased synthesis of unsaturated fatty acids at lowertemperatures (Yongmanitchai and Ward, Appl. Environ. Microbiol.57:419-25 (1991)). Based on process economics, this temperature shiftshould likely occur after the first phase of the two-stage fermentation,when the bulk of the organisms' growth has occurred.

It is contemplated that a variety of fermentation process designs may beapplied, where commercial production of omega fatty acids using theinstant Δ12 desaturase genes is desired. For example, commercialproduction of PUFAs from a recombinant microbial host may be produced bya batch, fed-batch or continuous fermentation process.

A batch fermentation process is a closed system wherein the mediacomposition is set at the beginning of the process and not subject tofurther additions beyond those required for maintenance of pH and oxygenlevel during the process. Thus, at the beginning of the culturingprocess the media is inoculated with the desired organism and growth ormetabolic activity is permitted to occur without adding additionalsubstrates (i.e., carbon and nitrogen sources) to the medium. In batchprocesses the metabolite and biomass compositions of the system changeconstantly up to the time the culture is terminated. In a typical batchprocess, cells moderate through a static lag phase to a high-growth logphase and finally to a stationary phase, wherein the growth rate isdiminished or halted. Left untreated, cells in the stationary phase willeventually die. A variation of the standard batch process is thefed-batch process, wherein the substrate is continually added to thefermentor over the course of the fermentation process. A fed-batchprocess is also suitable in the present invention. Fed-Batch processesare useful when catabolite repression is apt to inhibit the metabolismof the cells or where it is desirable to have limited amounts ofsubstrate in the media at any one time. Measurement of the substrateconcentration in fed-batch systems is difficult and therefore may beestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen and the partial pressure of waste gases (e.g., CO₂).Batch and fed-batch culturing methods are common and well known in theart and examples may be found in Thomas D. Brock in Biotechnology: ATextbook of Industrial Microbiology, 2^(nd) ed., (1989) SinauerAssociates: Sunderland, Mass.; or Deshpande, Mukund V., Appl. Biochem.Biotechnol., 36:227 (1992), herein incorporated by reference.

Commercial production of omega fatty acids using the instant Δ12desaturases may also be accomplished by a continuous fermentationprocess wherein a defined media is continuously added to a bioreactorwhile an equal amount of culture volume is removed simultaneously forproduct recovery. Continuous cultures generally maintain the cells inthe log phase of growth at a constant cell density. Continuous orsemi-continuous culture methods permit the modulation of one factor orany number of factors that affect cell growth or end productconcentration. For example, one approach may limit the carbon source andallow all other parameters to moderate metabolism. In other systems, anumber of factors affecting growth may be altered continuously while thecell concentration, measured by media turbidity, is kept constant.Continuous systems strive to maintain steady state growth and thus thecell growth rate must be balanced against cell loss due to media beingdrawn off the culture. Methods of modulating nutrients and growthfactors for continuous culture processes, as well as techniques formaximizing the rate of product formation, are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

Purification of PUFAs

The PUFAs may be found in the host microorganism as free fatty acids orin esterified forms such as acylglycerols, phospholipids, sulfolipids orglycolipids, and may be extracted from the host cell through a varietyof means well-known in the art. One review of extraction techniques,quality analysis and acceptability standards for yeast lipids is that ofZ. Jacobs (Critical Reviews in Biotechnology 12(5/6):463-491 (1992)). Abrief review of downstream processing is also available by A. Singh andO. Ward (Adv. Appl. Microbiol. 45:271-312 (1997)).

In general, means for the purification of PUFAs may include extractionwith organic solvents, sonication, supercritical fluid extraction (e.g.,using carbon dioxide), saponification, and physical means such aspresses, or combinations thereof. Of particular interest is extractionwith methanol and chloroform in the presence of water (E. G. Bligh & W.J. Dyer, Can. J. Biochem. Physiol. 37:911-917 (1959)). Where desirable,the aqueous layer can be acidified to protonate negatively-chargedmoieties and thereby increase partitioning of desired products into theorganic layer. After extraction, the organic solvents can be removed byevaporation under a stream of nitrogen. When isolated in conjugatedforms, the products may be enzymatically or chemically cleaved torelease the free fatty acid or a less complex conjugate of interest, andcan then be subject to further manipulations to produce a desired endproduct. Desirably, conjugated forms of fatty acids are cleaved withpotassium hydroxide.

If further purification is necessary, standard methods can be employed.Such methods may include extraction, treatment with urea, fractionalcrystallization, HPLC, fractional distillation, silica gelchromatography, high-speed centrifugation or distillation, orcombinations of these techniques. Protection of reactive groups, such asthe acid or alkenyl groups, may be done at any step through knowntechniques (e.g., alkylation or iodination). Methods used includemethylation of the fatty acids to produce methyl esters. Similarly,protecting groups may be removed at any step. Desirably, purification offractions containing GLA, STA, ARA, DHA and EPA may be accomplished bytreatment with urea and/or fractional distillation.

DESCRIPTION OF PREFERRED EMBODIMENTS

The ultimate goal of the work described herein is the development of anoleaginous yeast that accumulates oils enriched in ω-3 and/or ω-6 PUFAs.Toward this end, desaturases must be identified that functionefficiently in oleaginous yeast, to enable synthesis and highaccumulation of preferred PUFAs in these hosts. Identification ofefficient desaturases is also necessary for the manipulation of theratio of ω-3 to ω-6 PUFAs produced in host cells.

In previous work, the Applicants have isolated the native Yarrowialipolytica Δ12 desaturase and over-expressed this protein, resulting inincreased conversion of oleic acid to LA with respect to the wildtypecells (U.S. patent application Ser. No. 10/840,325, incorporatedentirely by reference; see also Example 2 herein and SEQ ID NOs:51 and52). Specifically, the percent substrate conversion (measured as([18:2]/[18:1+18:2])*100) was 74% in the transformed cells, as opposedto a percent substrate conversion of only 59% in wildtype Yarrowia.Despite the observed increase in LA availability within these hostcells, however, it was desirable to identify genes encoding proteinswith Δ12 desaturase activity that would enable even greater conversionof oleic acid to LA. This would permit increased availability of LA as asubstrate for high-level synthesis of a variety of ω-3 and/or ω-6 PUFAswithin the Y. lipolytica transformant cells (e.g., GLA, DGLA, ARA, ALA,STA, ETA, EPA, DPA and DHA). Thus, expression of a heterologous proteinhaving high-level Δ12 desaturase activity was therefore advantageous inthe pathway engineering of the organism.

To achieve these goals, in the present invention Applicants haveisolated and cloned a DNA fragment from Fusarium moniliforme thatencodes a Δ12 desaturase enzyme (SEQ ID NOs:3 and 4). Confirmation ofthis gene's activity as a Δ12 desaturase was provided based upon: 1.)restoration of LA biosynthesis (via complementation) upon expression ofthe Fusarium moniliforme gene in a Yarrowia lipolytica strain in whichthe native Δ12 desaturase gene was disrupted; and 2.) over-production ofLA in wild type Y. lipolytica cells expressing the F. moniliforme gene(Example 6).

The experimentation described above led to a surprising discovery,however, wherein the F. moniliforme Δ12 desaturase was more efficientthan the Yarrowia lipolytica Δ12 desaturase in producing 18:2 inYarrowia lipolytica (see Example 6, Table 11). Specifically, expressionof the F. moniliforme Δ12 desaturase under the control of the TEFpromoter in Yarrowia lipolytica was determined to produce higher levelsof 18:2 (68% product accumulation of LA) than were previously attainableby expression of a chimeric gene encoding the Yarrowia lipolytica Δ12desaturase under the control of the TEF promoter (59% productaccumulation of LA). This corresponds to a difference in percentsubstrate conversion (calculated as ([18:2]/[18:1+18:2])*100) of 85%versus 74%, respectively. Furthermore, the F. moniliforme Δ12 desaturasefunctioned much more efficiently than previous reports for any known Δ12desaturase (e.g., only 68% substrate conversion of oleic acid to 18:2was achieved upon overexpression of the Neurospora crassa Δ12 desaturasein S. cerevisiae [WO 2003/099216]).

On the basis of these results, expression of the present fungal F.moniliforme Δ12 desaturase is preferred relative to other known Δ12desaturases as a means to engineer an oleaginous yeast that accumulatesoils enriched in ω-3 and/or ω-6 PUFAs (however, one skilled in the artwould expect that the activity of the F. moniliforme Δ12 desaturasecould be enhanced in Yarrowia lipolytica, following e.g.,codon-optimization).

Additionally, Applicants have also identified a suite of Δ12 desaturasesorthologous to the Fusarium moniliforme protein described above fromAspergillus nidulans, Magnaporthe grisea, Neurospora crassa, Fusariumgraminearium, Aspergillus fumigatus and Aspergillus flavus (i.e., SEQ IDNOs:8, 12, 16, 20, 21 and 22). These proteins (including the Fusariummoniliforme Δ12 desaturase (SEQ ID NO:4)) clustered within a distinctsub-family of proteins (referred to herein as “Sub-family 2”) that arewell-distinguished from the proteins clustered within “Sub-family 1”(i.e., SEQ ID NOs:2, 6, 10,14 and 18, identified in co-pending U.S.Provisional Application 60/519,191 as Δ15 desaturases), despite allproteins' identification as homologous to the Y. lipolytica Δ12desaturase identified herein as SEQ ID NO:52 (characterized inco-pending U.S. patent application Ser. No. 10/840,325). Together, theproteins of sub-family 2 (identified herein as Δ12 desaturases andsupported by the functional characterization of the Neurospora crassaprotein as a Δ12 desaturase in WO 03/099216) represent a group ofproteins having at least 56.3% identity to one another (Example 4) andthey are well-distinguished by sequence homology from previouslydescribed Δ12 desaturases.

It is expected that this unique class of fungal Δ12 desaturases will beuseful for expression in oleaginous yeast (e.g., Yarrowia lipolytica) asa means to alter the fatty acid composition, based on the expectationthat they will function with high efficiency (i.e., percent substrateconversion, wherein % substrate conversion of oleic acid to LA of atleast about 70% is preferred, while a % substrate conversion to LA of atleast about 80% is particularly suitable, and a % substrate conversionto LA of at least about 85% is most preferred). Thus, one embodiment ofthe invention is a method of altering fatty acid profiles in anoleaginous yeast, whereby a Δ12 desaturase protein of sub-family 2 isexpressed alone or in combination with other fatty acid biosyntheticgenes (e.g., a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ12desaturase, a Δ15 desaturase, a Δ17 desaturase, a Δ9 desaturase, a Δ8desaturase and/or an elongase).

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by: 1.) Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions; Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y. (1984); and 3.) Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following Examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds), American Society for Microbiology: Washington,D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology 2^(nd) ed., Sinauer Associates: Sunderland,Mass. (1989). All reagents, restriction enzymes and materials used forthe growth and maintenance of bacterial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.),GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified.

E. coli TOP10 cells and E. coli Electromax DH10B cells were obtainedfrom Invitrogen (Carlsbad, Calif.). Max Efficiency competent cells of E.coli DH5a were obtained from GIBCO/BRL (Gaithersburg, Md.). E. coli(XL1-Blue) competent cells were purchased from the Stratagene Company(San Diego, Calif.). All E. coli strains were typically grown at 37° C.on Luria Bertani (LB) plates.

General molecular cloning was performed according to standard methods(Sambrook et al., supra). Oligonucleotides were synthesized bySigma-Genosys (Spring, Tex.). PCR products were cloned into Promega'spGEM-T-easy vector (Madison, Wis.).

DNA sequence was generated on an ABI Automatic sequencer using dyeterminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using acombination of vector and insert-specific primers. Sequence editing wasperformed in Sequencher (Gene Codes Corporation, Ann Arbor, Mich.). Allsequences represent coverage at least two times in both directions.Comparisons of genetic sequences were accomplished using DNASTARsoftware (DNASTAR Inc., Madison, Wis.).

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μl” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s) and “kB” means kilobase(s).

Cultivation of Yarrowia lipolytica

Yarrowia lipolytica strains ATCC #76982 and ATCC #90812 were purchasedfrom the American Type Culture Collection (Rockville, Md.). Y.lipolytica strains were usually grown at 28° C. on YPD agar (1% yeastextract, 2% bactopeptone, 2% glucose, 2% agar). For transformationselection, minimal medium (0.17% yeast nitrogen base (DIFCOLaboratories, Detroit, Mich.) without ammonium sulfate and without aminoacids, 2% glucose, 0.1% proline, pH 6.1) was used. Supplements ofadenine, leucine, lysine and/or uracil were added as appropriate to afinal concentration of 0.01%.

Fatty Acid Analysis of Yarrowia lipolytica

For fatty acid analysis, cells were collected by centrifugation andlipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can.J. Biochem. Physiol. 37:911-917 (1959)). Fatty acid methyl esters wereprepared by transesterification of the lipid extract with sodiummethoxide (Roughan, G., and Nishida I. Arch Biochem Biophys. 276(1):3846(1990)) and subsequently analyzed with a Hewlett-Packard 6890 GC fittedwith a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oventemperature was from 170° C. (25 min hold) to 185° C. at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) washarvested, washed once in distilled water, and dried under vacuum in aSpeed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) was added to thesample, and then the sample was vortexed and rocked for 20 min. Afteradding 3 drops of 1 M NaCl and 400 μl hexane, the sample was vortexedand spun. The upper layer was removed and analyzed by GC as describedabove.

Example 1 Construction of Yarrowia Expression Vectors

The present Example describes the construction of plasmid pY5, pY5-13(comprising a chimeric TEF promoter::XPR terminator gene), and pY5-20(comprising a chimeric hygromycin resistance gene).

Construction of Plasmid pY5

The plasmid pY5, a derivative of pINA532 (a gift from Dr. ClaudeGaillardin, Insitut National Agronomics, Centre de biotechnologieAgro-Industrielle, laboratoire de Genetique Moleculaire et CellularieINRA-CNRS, F-78850 Thiverval-Grignon, France), was constructed forexpression of heterologous genes in Yarrowia lipolytica (FIG. 3).

First, the partially-digested 3598 bp EcoRI fragment containing theARS18 sequence and LEU2 gene of pINA532 was subcloned into the EcoRIsite of pBluescript (Strategene, San Diego, Calif.) to generate pY2. TheTEF promoter (Muller S., et al. Yeast, 14: 1267-1283 (1998)) wasamplified from Yarrowia lipolytica genomic DNA by PCR using TEF5′ (SEQID NO:23) and TEF3′ (SEQ ID NO:24) as primers. PCR amplification wascarried out in a 50 μl total volume containing: 100 ng Yarrowia genomicDNA, PCR buffer containing 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl(pH 8.75), 2 mM MgSO₄, 0.1% Triton X-100, 100 μg/mL BSA (finalconcentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmoleof each primer and 1 μl of PfuTurbo DNA polymerase (Stratagene, SanDiego, Calif.). Amplification was carried out as follows: initialdenaturation at 95° C. for 3 min, followed by 35 cycles of thefollowing: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 1 min. Afinal extension cycle of 72° C. for 10 min was carried out, followed byreaction termination at 4° C. The 418 bp PCR product was ligated intopCR-Blunt to generate pIP-tef. The BamHI/EcoRV fragment of pIP-tef wassubcloned into the BamHI/SmaI sites of pY2 to generate pY4.

The XPR2 transcriptional terminator was amplified by PCR using pINA532as template and XPR5′ (SEQ ID NO:25) and XPR3′ (SEQ ID NO:26) asprimers. The PCR amplification was carried out in a 50 μl total volume,using the components and conditions described above. The 179 bp PCRproduct was digested with SacII and then ligated into the SacII site ofpY4 to generate pY5. Thus, pY5 (shown in FIG. 3) is useful as aYarrowia-E. coli shuttle plasmid containing:

-   -   1.) a Yarrowia autonomous replication sequence (ARS18);    -   2.) a ColE1 plasmid origin of replication;    -   3.) an ampicillin-resistance gene (AmpR), for selection in E.        coli;    -   4.) a Yarrowia LEU2 gene, for selection in Yarrowia;    -   5.) the translation elongation promoter (TEF P), for expression        of heterologous genes in Yarrowia; and    -   6.) the extracellular protease gene terminator (XPR2) for        transcriptional termination of heterologous gene expression in        Yarrowia.        Construction of Plasmids PY5-13 and pY5-20

pY5-13 and pY5-20 were constructed as derivatives of pY5 to faciliatesubcloning and heterologous gene expression in Yarrowia lipolytica.

Specifically, pY5-13 was constructed by 6 rounds of site-directedmutagenesis using pY5 as template. Both SalI and ClaI sites wereeliminated from pY5 by site-directed mutagenesis using oligonucleotidesYL5 and YL6 (SEQ ID NOs:27 and 28) to generate pY5-5. A SalI site wasintroduced into pY5-5 between the Leu2 gene and the TEF promoter bysite-directed mutagenesis using oligonucleotides YL9 and YL10 (SEQ IDNOs:29 and 30) to generate pY5-6. A PacI site was introduced into pY5-6between the LEU2 gene and ARS18 using oligonucleotides YL7 and YL8 (SEQID NOs:31 and 32) to generate pY5-8. An NcoI site was introduced intopY5-8 around the translation start codon of the TEF promoter usingoligonucleotides YL3 and YL4 (SEQ ID NOs:33 and 34) to generate pY5-9.The NcoI site inside the Leu2 gene of pY5-9 was eliminated using YL1 andYL2 oligonucleotides (SEQ ID NOs:35 and 36) to generate pY5-12. Finally,a BsiWI site was introduced into pY5-12 between the ColEI and XPR regionusing oligonucleotides YL61 and YL62 (SEQ ID NOs:37 and 38) to generatepY5-13.

Plasmid pY5-20 is a derivative of pY5. It was constructed by inserting aNot I fragment containing a chimeric hygromycin resistance gene into theNot I site of pY5. The chimeric gene had the hygromycin resistance ORFunder the control of the Yarrowia lipolytica TEF promoter.

Example 2 Cloning of the Yarrowia lipolytica Δ12 Desaturase andDisruption of the Endogenous Δ12 Desaturase Gene

Based on the fatty acid composition of Yarrowia lipolytica (ATCC #76982)which demonstrated that the organism could make LA (18:2) but not ALA(18:3), it was assumed that Y. lipolytica would likely contain gene(s)having Δ12 desaturase activity but not Δ15 desaturase activity. Thus,the present Example describes the use of degenerate PCR primers toisolate a partial coding sequence of the Yarrowia lipolytica Δ12desaturase, the use of the partial sequence to disrupt the native genein Yarrowia lipolytica, and subsequent cloning of the full-length gene.

Cloning of a Partial Putative Δ12 Desaturase Sequence from Yarrowialipolytica by PCR Using Degenerate PCR Primers

Genomic DNA was isolated from Yarrowia lipolytica (ATCC #76982) usingDNeasy Tissue Kit (Qiagen, Catalog #69504) and resuspended in kit bufferAE at a DNA concentration of 0.5 μg/μl. PCR amplifications wereperformed using the genomic DNA as template and several sets ofdegenerate primers made to amino acid sequences conserved betweendifferent Δ12 desaturases. The best results were obtained with a set ofupper and lower degenerate primers, P73 and P76, respectively, as shownin the Table below.

TABLE 4 Degenerate Primers Used For Amplification Of A Partial PutativeΔ12 Desaturase Degenerate Corresponding Primer Nucleotide Amino Acid SetDescription Sequence Sequence P73 (32) 5′-TGGGTCCTGGGCCA WVLGHECGH26-mers YGARTGYGGNCA-3′ (SEQ ID NO:40) (SEQ ID NO:39) P76 (64)5′-GGTGGCCTCCTCGGC (M/I)PFVHAEEAT 30-mers GTGRTARAANGGNAT-3′ (SEQ IDNO:42) (SEQ ID NO:41) [Note: Abbreviations are standard for nucleotidesand proteins. The nucleic acid degereracy code used is as follows: R =A/G; Y = C/T; and N = A/C/G/T.]

The PCR was carried out in an Eppendorf Mastercycler Gradientthermocycler according to the manufacturer's recommendations.Amplification was carried out as follows: initial denaturation at 95° C.for 1 min, followed by 30 cycles of denaturation at 95° C. for 30 sec,annealing at 58° C. for 1 min, and elongation at 72° C. for 1 min. Afinal elongation cycle at 72° C. for 10 min was carried out, followed byreaction termination at 4° C.

The expected (ca. 740 bp) size PCR product was detected by agarose gelelectrophoresis, isolated, purified, cloned into a pTA vector(Invitrogen), and sequenced. The resultant sequence had homology toknown Δ12 desaturases, based on BLAST program analysis (Basic LocalAlignment Search Tool; Altschul, S. F., et al., J. Mol. Biol.215:403-410 (1993)).

Targeted Disruption of Yarrowia lipolytica Δ12 Desaturase Gene

Targeted disruption of the Δ12 desaturase gene in Yarrowia lipolyticaATCC #76982 was carried out by homologous recombination-mediatedreplacement of the Δ12 desaturase gene with a targeting cassettedesignated as pY23D12. pY23D12 was derived from plasmid pY20 (Example1).

Specifically, pY23D12 was created by inserting a Hind III/Eco RIfragment into similarly linearized pY20. This 642 bp fragment consistedof (in 5′ to 3′ orientation): 3′ homologous sequence from position +718to +1031 (of the coding sequence (ORF) in SEQ ID NO:51), a Bgl IIrestriction site, and 5′ homologous sequence from position +403 to +717(of the coding sequence (ORF) in SEQ ID NO:51). The fragment wasprepared by PCR amplification of 3′ and 5′ sequences from the 642 bp PCRproduct using sets of PCR primers P99 and P100 (SEQ ID NOs:43 and 44)and P101 and P102 (SEQ ID NOs:45 and 46), respectively.

pY23D12 was linearized by Bgl II restriction digestion and transformedinto mid-log phase Y. lipolytica ATCC #76982 cells by the lithiumacetate method according to the method of Chen, D. C. et al. (ApplMicrobiol Biotechnol. 48(2):232-235 (1997)). Briefly, Y. lipolytica wasstreaked onto a YPD plate and grown at 30° C. for approximately 18 hr.Several large loopfuls of cells were scraped from the plate andresuspended in 1 mL of transformation buffer containing: 2.25 mL of 50%PEG, average MW 3350; 0.125 mL of 2 M Li acetate, pH 6.0; 0.125 mL of 2M DTT; and 50 μg sheared salmon sperm DNA.

About 500 ng of plasmid DNA was incubated in 100 μl of resuspendedcells, and maintained at 39° C. for 1 hr with vortex mixing at 15 minintervals. The cells were plated onto YPD hygromycin selection platesand maintained at 30° C. for 2 to 3 days.

Four hygromycin-resistant colonies were isolated and screened fortargeted disruption by PCR. One set of PCR primers (P119 [SEQ ID NO:47]and P120 [SEQ ID NO:48]) was designed to amplify a specific junctionfragment following homologous recombination. Another set of PCR primers(P121 [SEQ ID NO:49] and P122 [SEQ ID NO:50]) was designed to detect thenative gene. Three of the four hygromycin-resistant colonies werepositive for the junction fragment and negative for the native fragment,thus confirming targeted integration.

Determination of Fatty Acid Profile in the Δ12 Desaturase-DisruptedStrain

Disruption of the native Δ12 desaturase gene was further confirmed by GCanalysis of the total lipids in one of the disrupted strains, designatedas Q-d12D. Single colonies of wild type (ATCC #76982) and Q-d12D wereeach grown in 3 mL minimal media (formulation/L: 20 g glucose, 1.7 gyeast nitrogen base, 1 g L-proline, 0.1 g L-adenine, 0.1 g L-lysine, pH6.1) at 30° C. to an OD₆₀₀˜1.0. The cells were harvested, washed indistilled water, speed vacuum dried and subjected to directtrans-esterification and GC analysis (as described in the GeneralMethods).

The fatty acid profile of wildtype Yarrowia and the transformant Q-d12Dcomprising the disrupted Δ12 desaturase are shown below in Table 5.Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid),18:0,18:1 (oleic acid) and 18:2 (LA) and the composition of each ispresented as a % of the total fatty acids.

TABLE 5 Fatty Acid Composition (% Of Total Fatty Acids) In Wildtype AndTransformant Yarrowia lipolytica Strain 16:0 16:1 18:0 18:1 18:2 Wildtype 11 14 2 33 34 Q-d12D disrupted 6 15 1 74 nd *nd = not detectableResults indicated that the native Δ12 desaturase gene in the Q-d12Dstrain was inactivated. Thus, it was possible to conclude that there wasonly one gene encoding a functional Δ12 desaturase in Yarrowialipolytica ATCC #76982.Plasmid Rescue of the Yarrowia lipolytica Δ12 Desaturase Gene

Since the Δ12 desaturase gene was disrupted by the insertion of theentire pY23D12 vector that also contained an E. coliampicillin-resistant gene and E. coli ori, it was possible to rescue theflanking sequences in E. coli. For this, genomic DNA of Yarrowialipolytica strain Q-d12D was isolated using the DNeasy Tissue Kit.Specifically, 10 μg of the genomic DNA was digested with 50 μl ofrestriction enzymes Age I, Avr II, Nhe I and Sph I in a reaction volumeof 200 μl. Digested DNA was extracted with phenol:chloroform andresuspended in 40 μl deionized water. The digested DNA (10 μl) wasself-ligated in 200 μl ligation mixture containing 3 U T4 DNA ligase.Ligation was carried out at 16° C. for 12 hrs. The ligated DNA wasextracted with phenol:chloroform and resuspended in 40 μl deionizedwater. Finally, 1 μl of the resuspended ligated DNA was used totransform E. coli by electroporation and plated onto LB platescontaining ampicillin (Ap). Ap-resistant colonies were isolated andanalyzed for the presence of plasmids by miniprep. The following insertsizes were found in the recovered or rescued plasmids (Table 6):

TABLE 6 Insert Sizes Of Recovered Plasmids, According To RestrictionEnzyme Enzyme plasmid insert size (kB) Agel 1.6 Avrll 2.5 Nhel 9.4 Sphl6.6Sequencing of the plasmids was initiated with sequencing primers P99(SEQ ID NO:43) and P102 (SEQ ID NO:46).

Based on the sequencing results, a full-length gene encoding theYarrowia lipolytica Δ12 desaturase gene was assembled (1936 bp; SEQ IDNO:51). Specifically, the sequence encoded an open reading frame of 1257bases (nucleotides +283 to +1539 of SEQ ID NO:51), while the deducedamino acid sequence was 419 residues in length (SEQ ID NO:52). This genewas also also publically disclosed as YALI-CDS3053.1 within the publicY. lipolytica protein database of the “Yeast project Genolevures”(Center for Bioinformatics, LaBRI, Talence Cedex, France) (see alsoDujon, B. et al., Nature 430 (6995):35-44 (2004)).

Example 3 Expression of Yarrowia lipolytica Δ12 Desaturase ORF Under theControl of a Heterologous Yarrowia Promoter

The present Example describes the expression of the Y. lipolytica Δ12desaturase ORF (from Example 2) in a chimeric gene under the control ofa heterologous (non-Δ12 desaturase) Yarrowia promoter. This enabledcomplementation of the Δ12 desaturase-disrupted mutant andoverproduction of LA in the wildtype Y. lipolytica strain.

Specifically, the ORF encoding the Y. lipolytica Δ12 desaturase was PCRamplified using upper primer P147 (SEQ ID NO:53) and lower primer P148(SEQ ID NO:54) from the genomic DNA of Y. lipolytica ATCC #76982. Thecorrect sized (1260 bp) fragment was isolated, purified, digested withNco I and Not I and cloned into NcoI-Not I cut pY5-13 vector (Example1), such that the gene was under the control of the TEF promoter.Correct transformants were confirmed by miniprep analysis and theresultant plasmid was designated pY25-d12d.

Plasmids pY5-13 (the “control”) and pY25-d12d were each individuallytransformed into Y. lipolytica ATCC #76982 wild-type (WT) andΔ12-disrupted strains (Q-d12D), using the transformation methoddescribed in Example 2. Positive transformants were selected on Bio101DOB/CSM-Leu plates.

Single colonies of transformants were grown up and GC analyzed asdescribed in the General Methods. Results are shown in the Table below.Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid),18:0,18:1 (oleic acid) and 18:2 (LA); and the composition of each ispresented as a % of the total fatty acids. “D12d SC” was calculatedaccording to the following formula: ([18:2]/[18:1+18:2])*100 andrepresents percent substrate conversion (SC).

TABLE 7 Fatty Acid Composition (% Of Total Fatty Acids) Of Yarrowialipolytica Transformed With Yarrowia lipolytica Δ12 Desaturase Gene % %% % Strain Plasmid % 16:0 16:1 18:0 18:1 18:2 D12d SC Q-d12D pY5-13 8 102 80 nd 0 Q-d12D pY25-d12d 11 8 2 34 45 57 WT pY5-13 10 10 1 32 47 59 WTpY25-d12d 12 7 2 21 59 74 *nd = not detectableThe results showed that expression of Y. lipolytica Δ12 desaturase usingthe TEF promoter restores production of 18:2 in the strain with theendogenous gene disrupted.

Additionally, the results demonstrated that overexpression of the Y.lipolytica Δ12 desaturase gene in wild type cells resulted in increasedlevels of LA production (18:2). Specifically, the % product accumulationof LA increased from 47% in wild type cells to 59% in transformant cells(representing a change in the percent substrate conversion [“D12d SC”]from 59% in the wild type to 74% in those cells transformed with thechimeric Δ12 desaturase).

Example 4 Identification of Δ12 Desaturases from Filamentous Fungi

The present Example describes the identification of Δ12 desaturases invarious filamentous fungi. These sequences were identified based ontheir homology to the Yarrowia lipolytica Δ12desaturase (Example 2);and, the sequences from each species fell into one of two “sub-families”based on phylogenetic analyses.

BLAST searches (Basic Local Alignment Search Tool; Altschul, S. F., etal., J. Mol. Biol. 215:403410 (1993)) were conducted using the Yarrowialipolytica Δ12 desaturase protein sequence (SEQ ID NO:52) as a queryagainst available sequence databases of filamentous fungi, including:1.) public databases of Neurospora crassa, Magnaporthe grisea,Aspergillus nidulans and Fusarium graminearium; and 2.) a DuPont ESTlibrary of Fusarium moniliforme strain M-8114 (E.I. du Pont de Nemoursand Co., Inc., Wilmington, Del.) (F. moniliforme strain M-8114 availablefrom the Fusarium Research Center, University Park, Pa.; see also PlantDisease 81(2): 211-216 (1997)). These BLAST searches resulted in theidentification of two homologs to the Yarrowia lipolytica Δ12 desaturaseprotein within each organism. The Table below summarizes detailsconcerning each of these homologs.

TABLE 8 Description Of ORFs Having Homology To The Yarrowia lipolyticaΔ12 Desaturase SEQ ID NOs* Source Abbreviation Organism 1, 2 ESTsequence database, E. I. Fm1 or Fusarium duPont de Nemours and Co., Inc.Fm d15 moniliforme 3, 4 EST sequence database, E. I. Fm2 or FusariumduPont de Nemours and Co., Inc. Fm d12 moniliforme 5, 6 Contig 1.122(scaffold 9) in the A. nidulans An1 or Aspergillus genome project(sponsored An d15 nidulans by the Center for Genome Research (CGR),Cambridge, MA); see also WO 2003/099216 7, 8 Contig 1.15 (scaffold 1) inthe A. nidulans An2 or Aspergillus genome project; An d12 nidulansAAG36933 9, 10 Locus MG08474.1 in contig 2.1597 Mg1 or Magnaporthe inthe M. grisea genome project Mg d15 grisea (sponsored by the CGR andInternational Rice Blast Genome Consortium) 11, Locus MG01985.1 incontig 2.375 in Mg2 or Magnaporthe 12 the M. grisea genome project Mgd12 grisea 13, GenBank Accession No. Nc1 or Neurospora 14 AABX01000577);see also WO Nc d15 crassa 2003/099216 15, GenBank Accession No. Nc2 orNeurospora 16 AABX01000374; see also WO Nc d12 crassa 2003/099216 17,Contig 1.320 in the F. graminearium Fg1 or Fusarium 18 genome project(sponsored by the Fg d15 graminearium CGR and the InternationalGibberella zeae Genomics Consortium (IGGR); BAA33772.1) 19, Contig 1.233in the F. graminearium Fg2 or Fusarium 20 genome project Fg d12graminearium *Note: Odd SEQ ID NOs refer to ORF nucleotide sequences andeven SEQ ID NOs refer to the deduced amino acid sequences.All of the homologs were either unannotated or annotated as a Δ12desaturase or fatty acid desaturase. Furthermore, the nucleotidesequences from F. graminearium were genomic with putative intronsequences; the Applicants made a tentative assembly of the deduced aminoacids for comparison with amino acid sequences from the other homologs.

Phylogenetic tree analysis of the Δ12 desaturase homologs from eachspecies using the Megalign program of the LASERGENE bioinformaticscomputing suite (Windows 32 Megalign 5.06 1993-2003; DNASTAR Inc.,Madison, Wis.) unexpectedly revealed two sub-families. As shown in FIG.4, Nc1, Mg1, Fg1, Fm1 and An1 clustered in “sub-family 1” of theproteins having homology to the Yarrowia lipolytica Δ12 desaturase whileFg2, Fm2, Mg2, Nc2 and An2 clustered within “sub-family 2” of theYarrowia lipolytica Δ12 desaturase protein homologs.

Subsequently, a single Δ12 desaturase homolog was also identified inpublic sequence databases for two additional organisms, as shown belowin Table 9.

TABLE 9 Additional ORFs Having Homology To The Yarrowia lipolytica Δ12Desaturase SEQ ID NO Source Symbol Organism 21 AFA.133c 344248:345586reverse Af d12 Aspergillus (AfA5C5.001c) in the Aspergillus fumigatusfumigatus genome project (sponsored by Sanger Institute, collaboratorsat the University of Manchester, and The Institute of Genome Research(TIGR)) 22 GenBank Accession No. AY280867 — Aspergillus (VERSIONAY280867.1; flavus GI: 30721844)These additional sequences clustered within sub-family 2 of the Yarrowialipolytica Δ12desaturase protein homologs.

Each of the proteins having homology to the Yarrowia lipolyticaΔ12desaturase were then aligned using the method of Clustal W (slow,accurate, Gonnet option; Thompson et al., Nucleic Acids Res.22:4673-4680 (1994)) of the Megalign program of DNASTAR software. Thepercent identities revealed by this method were used to determinewhether the proteins were orthologs (FIG. 5). In addition, the homologswere compared to the known Δ12 desaturases shown below in Table 10 (FIG.6).

TABLE 10 Known Δ12 Desaturases Used In Clustal W Analysis FIG. 6 Ref.GenBank No. Symbol Description Accession No. 1 Sp d12 Spirulineplatensis Δ12 X86736 desaturase 2 Ce d12 Caenorhabditis elegans AF240777Δ12 desaturase 3 Cr d12 Chlamydomonas AB007640 reinhardtii Δ12desaturase 4 Cv d12 Chlorella vulgaris Δ12 AB075526 desaturase 5 AtMArabidopsis thaliana AP002063 d12 microsomal Δ12 desaturase 6 Yl d12Yarrowia lipolytica Δ12 YALI-CDS3053.1 desaturase (SEQ ID within thepublic Y. NO: 52 herein) lipolytica protein database of the “Yeastproject Genolevures” (Center for Bioinformatics, LaBRI, Talence Cedex,France) (see also Dujon, B. et al., Nature 430 (6995): 35-44 (2004));see also U.S. Patent Application No. 10/840325 7 Dh d12 Debaryomyceshansenii >Ctg0330-0000227-2.1 Δ12 desaturase (see “Yeast projectGenolevures”) 8 MaA Mortierella alpina Δ12 >gi|6448794|gb|AAF08684.1|d12 desaturase AF110509_1 9 Mr d12 Mucor rouxii Δ12 AF161219 desaturase10 Pa d12 Pichia augusta Δ12 >Ctg1334- desaturase 0000001-1.1. (see“Yeast project Genolevures”)

The proteins of sub-family 1 (SEQ ID NOs:2, 6, 10, 14 and 18; FIG. 5)were at least 46.2% identical to each other and were less than 45.2%identical to the proteins of sub-family 2 (SEQ ID NOs:4, 8, 12, 16, 20;FIG. 5). Furthermore, the proteins of sub-family 2 (SEQ ID NOs:4, 8, 12,16, 20; FIG. 5) were at least 56.3% identical to each other. The maximumidentity between the proteins of sub-family 2 and the Δ12 desaturasesfrom S. platensis, C. elegans, C. reinhardtii, C. vulgaris, A. thaliana,Y. lipolytica, D. hansenii, M. alpina, M. rouxii, and P. augusta is51.6% (FIG. 6).

Although not shown in FIG. 5 or FIG. 6, the Aspergillus flavus Δ12desaturase homolog (SEQ ID NO:22) was similarly compared to the otherproteins of sub-family 2 (SEQ ID NOs:4, 8, 12, 16, 20 and 21) using themethod of Clustal W (supra). The following results were obtained:

-   -   79% protein identity of Aspergillus flavus against A. fumigatus,        68.5% protein identity against N. crassa, 60.9% protein identity        against F. graminearium, 61.8% protein identity against F.        moniliforme, 66.1% protein identity against M. grisea and 76%        protein identity against A. nidulans. Thus, based on the        homologies in FIGS. 5 and 6 and those presented above, the F.        moniliforme Δ12desaturase (SEQ ID NO:4) was at least 56.3%        identical to the remaining Δ12 desaturase proteins of sub-family        2 (herein defined as SEQ ID NOs:4, 8, 12, 16, 20, 21 and 22),        based on the homology between the F. moniliforme and M. grisea        protein sequences.

The analyses above clearly differentiated the two sub-families ofproteins having homology to the Yarrowia lipolytica Δ12 desaturase (SEQID NO:52). Additionally, it was known that yeast such as Y. lipolyticacan only synthesize 18:2 (but not 18:3), while each of the fivefilamentous fungi are able to synthesize both 18:2 and 18:3.Furthermore, a single Δ12 desaturase was isolated from Yarrowia, whilemost of the fungi had two homologs to the Yarrowia Δ12 desaturase. Thus,the Applicants postulated that one of the sub-families of desaturases inthese organisms represented a Δ12 desaturase (permitting conversion ofoleic acid to LA (18:2)) and the other represented a Δ15 desaturase(permitting conversion of LA to ALA (18:3)).

Example 5 Construction of Expression Plasmid pY35 (TEF::Fm2), Comprisingthe Fusarium moniliforme Desaturase of Sub-Family 2 (Encoding a PutativeΔ12 Desaturase)

The present Example describes the construction of an expression plasmidcomprising the Fusarium moniliforme Δ12 desaturase of sub-family 2(“Fm2” or “Fm d12”) identified in Example 4. Specifically, a chimericgene was created, such that the putative Δ12 desaturase would beexpressed under the control of the Yarrowia TEF promoter. This wouldenable subsequent determination of the protein's activity in Yarrowialipolytica, by testing the ability of the expressed ORF to confer LAproduction in the wild type strain and to complement a Δ12desaturase-disrupted mutant (infra, Example 6).

The ORF encoding the F. moniliforme Δ12 desaturase was PCR amplifiedusing the cDNA clones ffm2c.pK007.g13 and ffm2c.pk001.p18 containing thefull-length cDNA as the template and using upper and lower primers P194(SEQ ID NO:55) and P195 (SEQ ID NO:56). The PCR was carried out in anEppendorf Mastercycler Gradient Cycler using TA Taq and pfu polymerases,per the manufacturer's recommendations. Amplification was carried out asfollows: initial denaturation at 95° C. for 1 min, followed by 30 cyclesof denaturation at 95° C. for 30 sec, annealing at 58° C. for 1 min, andelongation at 72° C. for 1 min. A final elongation cycle at 72° C. for10 min was carried out, followed by reaction termination at 4° C.

The correct-sized (ca. 1441 bp) fragment was obtained from bothtemplates. The fragment was purified from an agarose gel using a QiagenDNA purification kit, digested with NcoI and NotI and cloned into NcoIand NotI cut pY25-d12d (Example 3), thereby replacing the Yarrowia Δ12desaturase ORF in the TEF::YI D12d chimeric gene. This resulted increation of a 8571 bp plasmid designated as pY35, which contained aTEF::Fm2 chimeric gene (as opposed to the Yarrowia Δ12 desaturase).Plasmid pY35 additionally contained the E. coli origin of replication,the bacterial ampicillin resistance gene, Yarrowia Leu 2 gene and theYarrowia autonomous replication sequence (ARS).

Example 6 Expression of Plasmid DY35 (TEF::Fm2), Comprising the Fusariummoniliforme Desaturase of Sub-Family 2 (Encoding a Putative Δ12Desaturase) In Yarrowia lipolytica

The present Example describes expression of plasmid pY35 (comprising thechimeric TEF::Fm2 gene; from Example 5) in Yarrowia lipolytica.Specifically, the ability of the expressed F. moniliforme ORF comprisingthe putative Δ12 desaturase was tested for its ability to confer LAproduction in the wild type strain of Y. lipolyica and to complement theΔ12 desaturase-disrupted mutant (from Example 2).

Plasmids pY5 (vector alone control, from Example 1), pY25-d12d (TEF::YID12d, the “positive control” from Example 3), and pY35 (TEF::Fm2) wereeach individually transformed into wild type (WT) and Δ12desaturase-disrupted (Q-d12D) strains of Yarrowia lipolytica ATCC#76892, using the transformation procedure described in Example 2.Transformant cells were selected on Bio101 DOB/CSM-Leu plates.

Single colonies of wild type and transformant cells were each grown in 3mL minimal media, harvested, washed, dried and analyzed, as described inExample 2 and the General Methods.

The fatty acid profile of wildtype Yarrowia and each of thetransformants are shown below in Table 11 (results obtained from twoexperiments). Fatty acids are identified as 16:0 (palmitate), 16:1(palmitoleic acid), 18:0, 18:1 (oleic acid), 18:2 (LA) and 18:3 (ALA)and the composition of each is presented as a % of the total fattyacids. “d12d SC” was calculated according to the following formula:([18:2]/[18:1+18:2])*100 and represents percent substrate conversion to18:2.

TABLE 11 Confirmation Of The Fusarium moniliforme Fm2 ORF's Δ12Desaturase Activity When Expressed In Yarrowia lipolytica % % % % % %d12d strain 16:0 16:1 18:0 18:1 18:2 ALA % SC WT 11 11 2 33 39 0.0 55WT + pY5 9 25 0 32 35 0.0 52 (vector alone) WT + TEF::Fm2 12 5 1 12 680.4 85 Q-d12D 6 11 1 80 0 0.0 0 Q-d12D + TEF::Fm2 10 6 1 27 53 0.3 67

The results above demonstrated that the F. moniliforme ORF referred toherein as Fm2, and identified as a protein within sub-family 2 of thoseproteins having homology to the Yarrowia lipolytica Δ12 desaturase, is aΔ12 desaturase. Based on this confirmation, the Applicants predict thatall other members of sub-family 2 (SEQ ID NOs:8, 12, 16, 20, 21 and 22)also will have Δ12 desaturase functionality.

Additionally, the results demonstrated that, unexpectedly, the Fusariummoniliforme Δ12 desaturase (Fm2) is even more efficient in its activityin Yarrowia than the native Y. lipolytica Δ12 desaturase. Specifically,comparison of the data in Table 11 to that of Table 7 (Example 3)reveals a percent substrate conversion (SC) of 85% in WT+TEF::Fm2 cellsversus only 74% in WT+TEF::YI D12d cells. Likewise, the percentsubstrate conversion in Q-d12D+TEF::Fm2 cells was 67%, while the percentsubstrate conversion was only 57% in Q-d12D+TEF::YI D12d cells. Thus, itwould be expected that expression of the Fusarium moniliforme Δ12desaturase, in combination of other genes for PUFA biosynthesis (e.g., aΔ6 desaturase, elongase, Δ5 desaturase, Δ17 desaturase, Δ9 desaturase,Δ4 desaturase, Δ8 desaturase, Δ15 desaturase), would result in higherproduction of ω-3 and ω-6 PUFAs than would result using the native Y.lipolytica Δ12 desaturase.

Example 7 Production of DGLA in Yarrowia lipolytica Using Chimeric GenesConstructed with the Fusarium moniliforme Δ12 Desaturase

Construct pKUNF12T6E (FIG. 7; SEQ ID NO:57) was generated to integratefour chimeric genes (comprising the Fusarium moniliforme Δ12 desaturase,a Δ6 desaturase and 2 elongases) into the Ura3 loci of wild typeYarrowia strain ATCC #20362, to thereby enable production of DGLA. ThepKUNF12T6E plasmid contained the following components:

TABLE 12 Description of Plasmid pKUNF12T6E (SEQ ID NO: 57) RE Sites AndNucleotides Within SEQ ID NO: 57 Description Of Fragment And ChimericGene Components Ascl/BsiWl 784 bp 5′ part of Yarrowia Ura3 gene (GenBankAccession (9420-8629) No. AJ306421) Sphl/Pacl 516 bp 3′ part of YarrowiaUra3 gene (GenBank Accession (12128-1) No. AJ306421) Swal/BsiWlFBAIN::EL1S: Pex20, comprising: (6380-8629) FBAIN: FBAIN promoter (SEQID NO: 58; see co- pending U.S. Patent Application No. 60/519971,describing a fructose-bisphosphate aldolase enzyme promoter) EL1S:codon-optimized elongase 1 gene (SEQ ID NO: 59), derived fromMortierella alpina (GenBank Accession No. AX464731) Pex20: Pex20terminator sequence from Yarrowia Pex20 gene (GenBank Accession No.AF054613) Bglll/Swal TEF::Δ6S::Lip1, comprising: (4221-6380) TEF: TEFpromoter (GenBank Accession No. AF054508) Δ6S: codon-optimized Δ6desaturase gene (SEQ ID NO: 61), derived from Mortierella alpina(GenBank Accession No. AF465281) Lip1: Lip1 terminator sequence fromYarrowia Lip1 gene (GenBank Accession No. Z50020) Pmel/ClalFBA::F.Δ12::Lip2, comprising: (4207-1459) FBA: FBA promoter (SEQ ID NO:63; see co-pending U.S. Patent Application No. 60/519971 describing afructose-bisphosphate aldolase enzyme promoter) F.Δ12: Fusariummoniliforme Δ12 desaturase gene (SEQ ID NO: 3) Lip2: Lip2 terminatorsequence from Yarrowia Lip2 gene (GenBank Accession No. AJ012632)Clal/Pacl TEF::EL2S::XPR, comprising: (1459-1) TEF: TEF promoter(GenBank Accession No. AF054508) EL2S: codon-optimized elongase gene(SEQ ID NO: 64), derived from Thraustochytrium aureum (U.S. Pat. No.6,677,145) XPR: XPR terminator sequence of Yarrowia Xpr gene (GenBankAccession No. M17741)

The pKUNF12T6E plasmid was digested with AscI/SphI, and then used fortransformation of wild type Y. lipolytica ATCC #20362 (as described inExample 2). The transformant cells were plated onto5-fluorouracil-6-carboxylic acid monohydrate (“FOA”) selection mediaplates (0.17% yeast nitrogen base (DIFCO Laboratories, Detroit, Mich.)without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, 75mg/L uracil, 75 mg/L uridine, 20 g/L agar and 800 mg/L FOA (ZymoResearch Corp., Orange, Calif. 92867)) and maintained at 30° C. for 2 to3 days. The FOA resistant colonies were picked and streaked ontoselection plates comprising either minimal media (20 g/L glucose, 1.7g/L yeast nitrogen base without amino acids, 1 g/L L-proline, 0.1 g/LL-adenine, 0.1 g/L L-lysine, pH 6.1) or minimal media plus 0.01% uracil(“MMU”). The colonies that could grow on MMU plates but not on theminimal media plates were selected as Ura-strains. Single colonies ofUra-strains were then inoculated into liquid MMU at 30° C. and shaken at250 rpm/min for 2 days.

GC analyses (as described in the General Methods) showed the presence ofDGLA in the transformants containing the 4 chimeric genes of pKUNF12T6E,but not in the wild type Yarrowia control strain. Most of the selected32 Ura⁻ strains produced about 6% DGLA of total lipids. There were 2strains that produced about 8% DGLA of total lipids.

Example 8 Synthesis of a Codon-Optimized Δ12 Desaturase Gene forExpression in Yarrowia lipolytica

A codon-optimized Δ12 desaturase gene will be designed, based on theFusarium moniliforme DNA sequence (SEQ ID NO:3), according to theYarrowia codon usage pattern, the consensus sequence around the ATGtranslation initiation codon, and the general rules of RNA stability(Guhaniyogi, G. and J. Brewer, Gene 265(1-2):11-23 (2001)).

Determining the Preferred Codon Usage in Yarrowia lipolytica

Codon usage in Yarrowia lipolytica is shown in Table 13. It is based onthe coding region of approximately 100 genes of Y. lipolytica found inthe National Center for Biotechnology Information public database. Thecoding regions of these genes, comprising 121, 167 bp, were translatedby the Editseq program of DNAStar to the corresponding 40,389 aminoacids and were tabulated to determine the Y. lipolytica codon usageprofile shown in Table 13. The column titled “No.” refers to the numberof times a given codon encodes a particular amino acid in the sample of40,389 amino acids. The column titled “%” refers to the frequency that agiven codon encodes a particular amino acid. Entries shown in bold textrepresent the codons favored in Yarrowia lipolytica.

TABLE 13 Codon Usage In Yarrowia lipolytica Amino Codon Acid No. % GCAAla (A) 359 11.4 GCC Ala (A) 1523 48.1 GCG Ala (A) 256 8.1 GCU Ala (A)1023 32.3 AGA Arg (R) 263 13.2 AGG Arg (R) 91 4.6 CGA Arg (R) 1133 56.8CGC Arg (R) 108 5.4 CGG Arg (R) 209 1.0 CGU Arg (R) 189 9.5 AAC Ans (N)1336 84.0 AAU Ans (N) 255 16.0 GAC Asp (D) 1602 66.8 GAU Asp (D) 79533.2 UGC Cys (C) 268 53.2 UGU Cys (C) 236 46.8 CAA Gln (Q) 307 17.0 CAGGln (Q) 1490 83.0 GAA Glu (E) 566 23.0 GAG Glu (E) 1893 77.0 GGA Gly (G)856 29.7 GGC Gly (G) 986 34.2 GGG Gly (G) 148 5.1 GGU Gly (G) 893 31.0CAC His (H) 618 65.5 CAU His (H) 326 34.5 AUA Ile (I) 42 2.1 AUC Ile (I)1106 53.7 AUU Ile (I) 910 44.2 CUA Leu (L) 166 4.7 CUC Leu (L) 1029 29.1CUG Leu (L) 1379 38.9 CUU Leu (L) 591 16.7 UUA Leu (L) 54 1.5 UUG Leu(L) 323 9.1 AAA Lys (K) 344 14.8 AAG Lys (K) 1987 85.2 AUG Met (M) 1002100 UUC Phe (F) 996 61.1 UUU Phe (F) 621 38.9 CCA Pro (P) 207 9.6 CCCPro (P) 1125 52.0 CCG Pro (P) 176 8.2 CCU Pro (P) 655 30.2 AGC Ser (S)335 11.3 AGU Ser (S) 201 6.8 UCA Ser (5) 221 7.5 UCC Ser (S) 930 31.5UCG Ser (S) 488 16.5 UCU Ser (S) 779 26.4 UAA Term 38 46.9 UAG Term 3037.0 UGA Term 13 16.1 ACA Thr (T) 306 12.7 ACC Thr (T) 1245 51.6 ACG Thr(T) 269 11.1 ACU Thr (T) 595 24.6 UGG Trp (W) 488 100 UAC Tyr (Y) 98883.2 UAU Tyr (Y) 200 16.8 GUA Val (V) 118 4.2 GUC Val (V) 1052 37.3 GUGVal (V) 948 33.6 GUU Val (V) 703 24.9The synthetic, codon-optimized Δ12 desaturase is made by methods knownto one skilled in the art.

For further optimization of gene expression in Y. lipolytica, theconsensus sequence around the ‘ATG’ initiation codon of 79 genes wasexamined. Seventy seven percent of the genes analyzed had an ‘A’ in the−3 position (with the first ‘A’ of the translation initiation codon(ATG) labeled as +1), indicating a strong preference for ‘A’ at thisposition. There was also preference for ‘A’ or ‘C’ at the −4, −2 and −1positions, an ‘A’, ‘C’ or ‘T’ at position +5, and a ‘G’ or ‘C’ atposition +6. Thus, the preferred consensus sequence of thecodon-optimized translation initiation site for optimal expression ofgenes in Y. lipolytica is ‘MAMMATGNHS’ (SEQ ID NO:66), wherein thenucleic acid degeneracy code used is as follows: M=A/C; S=C/G; H=A/C/T;and N=A/C/G/T.

One skilled in the art would readily be able to use the informationprovided above to synthesize a codon-optimized Δ12 desaturase gene,based on the Fusarium moniliforme Δ12 desaturase sequence providedherein.

1. An isolated nucleic acid fragment encoding a fungal Δ12 desaturase enzyme, encoding the amino acid sequence as set forth in SEQ ID NO:4; or, an isolated nucleic acid fragment that is complementary to over the full length of the isolated nucleic acid sequence encoding the amino acid sequence as set forth in SEQ ID NO:4.
 2. The isolated nucleic acid fragment of claim 1 as set forth in SEQ ID NO:3.
 3. The isolated nucleic acid fragment of claim 1 isolated from Fusarium moniliforme.
 4. A chimeric gene comprising the isolated nucleic acid fragment of claim 1 operably linked to suitable regulatory sequences.
 5. A method for the production of linoleic acid comprising: (a) providing an oleaginous yeast comprising: (i) the isolated nucleic acid fragment according to claim 1; and (ii) a source of oleic acid; (b) growing the yeast of step (a) under conditions wherein the nucleic acid fragment encoding a polypeptide having Δ12 desaturase activity is expressed and the oleic acid is converted to linoleic acid; and (c) optionally recovering the linoleic acid of step (b).
 6. A method for the production of polyunsaturated fatty acids comprising: (a) providing an oleaginous yeast comprising: (i) the isolated nucleic acid fragment of claim 1; and (ii) genes encoding a functional ω-3/ω-6 fatty acid biosynthetic pathway; (b) providing a source of desaturase substrate comprising oleic acid; and (c) growing the oleaginous yeast of step (a) with the desaturase substrate of (b) under conditions wherein polyunsaturated fatty acids are produced; and (d) optionally recovering the polyunsaturated fatty acids of step (c). 